antibody titrations / controls
The missing sentence was: 'In addition a lot of antibodies also
bind on other cells and the amount of active antibody in the
bottle might vary. Therefore you should have approximately 10^3
fold excess.... Antigen concentration is indeed only of
relevance with respect to depletion. Regarding the variations in
the binding kinetics we have some applications where we want to
stay below 5% variation. The higher the excess of antibody,the
better. We use goat anti mouse*PE Fab2 at 12ug/ml and
have to run at least for 30 minutes to saturate and to keep
the day to day variations between the samples low. This
might be due to a lower affinity or the polyclonal nature of
the antibody. As most of our samples have a normal
distribution of antigen load, we estimate the degree of
saturation by the shape of the distribution in the
fluorescence histogram.
Gerhard Nebe-v.Caron,
Unilever Research, Colworth,
I just want to add my annual reminder that the concentration of
antibody to use in a staining mixture is essentially independent
of the number of cells being stained, but is completely
dependent on the stain volume! 1 ug of antibody is more than
enough for (in general) well over 10^8 cells expressing a
typical antigen--thus, it doesn't matter if you have 10^5, 10^6,
or 10^7 cells in your tube! Indeed, many antibodies will stain
equally well at 10^8 cells (in, for instance, 100 ul volume).
On the other hand, a reagent titred to be used in a 50ul stain
volume may not stain very well in 500 ul. Specifically: 1 ug
of antibody (IgG) is about 3.6 x 10^12 molecules--which (in
divalent fashion) binds 7.2x10^12 molecules of antigen. A
typical antigen may be present in concentrations of 10^4 to 10^5
per cell, meaning that 1 ug of antibody would be enough to stain
7.2 x 10^7 to 7.2 x 10^8 cells. And, if only 10% of the cells
express an antigen, you can multiply this by another factor of
10!
mario roederer, stanford
Actually, isotype controls are not a particularly good control.
They are rarely matched: the F/P ratio is not the same, and how
do you know if you are using them at exactly the same
concentration as the reagent of choice? If you don't know that
the F/P ratio is exactly the same, and if you don't know if you
are using it at exactly the same concentration as your antibody
reagent, then it isn't the right control. Indeed, since each
one of your commercial reagents is titrated by the manufacturer
to give optimal signal to background, each one is sold at a
different concentration of antibody. Have you contacted the
manufacturers to determine the bottled concentration of each
reagent you use, so that you can use the appropriate
concentration of your isotype control? And then use a different
isotype concentration as the control for each reagent in your
various panels? If the answer to either of these questions is
"no", then how can you assert that your isotype control actually
gives you the correct amount of background binding in your
experiment? i.e., your "isotype control" does no more than let
you that there may actually be some background binding, but
doesn't give you the ability to estimate how much. In fact,
I've known people to "titrate" their isotype controls to get
background binding that is less than what they think their
positive should be. Hmm. In another way, isotype controls are
rarely used properly: most people do a single sample that has
all isotype controls in all channels. This doesn't help! One
must use a control for which cells are stained with all reagents
EXCEPT the one of interest (and if you insist on using an
isotype control for that channel, so be it). We term these
controls "FMO" or "Fluorescence Minus One" controls. (For more
discussion of the need of FMO controls, see my paper on
Compensation in the upcoming issue of Cytometry). Staining
controls are very difficult to generate. In general, the best
control for antibody binding is a cell that is exactly the same
as your cell of interest, but lacking the antigen of interest.
Of course, this is rarely achievable. However, one will often
find very similar cells that meet the bill. In
immunophenotyping of peripheral blood, you can use
"nonexpressing" cell types as internal controls (i.e., naive T
cells can serve as a control for measuring activation markers on
memory T cells). Of course, you need to be careful, because
some "nonexpressing" cells actually express the marker. Isotype
controls have their place. However, most people don't use them
properly. In general, I counsel people NOT to use isotype
controls, but rather to use their brains to come up with a set
of appropriate negative controls (which MUST be included in all
experiments, as others have noted). Blind reliance on isotype
controls is one of the most common mistakes in publications--and
leads to the erroneous placement of gates. In any case, you are
correct that investigators need to be educated more. This is
one of the discussions that pops up every few years on the
mailing list; perhaps it's time to have a FAQ's page (no pun
intended!) on the Purdue site which includes the various
discussion points, and rather than coming up with a conclusion,
this page can simply serve to put forth various peoples' views
so that researchers can judge for themselves whether or not
isotype controls are useful. mr (PS, there is no such thing as
"bad data", only "bad interpretation of data.").
mario roederer, stanford
Yes... much of what you say would be true... if the use of
isotype controls were scientific. But, as has been endlessly
discussed on this list, they are NOT. (See also: O'Gorman MRG,
Thomas JA: Isotype Controls-Time to Let Go?; Cytometry 38:7880, 1999.) Proper science is to use a control (compared to a
test sample) in which only a single variable has been changed.
not), there are several more variables in your experiment. (1)
The concentration of the isotype. If you have not matched the
concentration of the isotype control to each of your antibodies
(and each antibody, incidentally, is used at a different
concentration, so you better be using isotypes at different
concentrations for each antibody you are controlling), then the
isotype control is no longer a control, but another test
sample. (2) The Fluor:Protein ratio (F/P) of the isotype. If
the F/P ratio is different than the test antibody (and how are
you ever going to know this, unless you make both antibody and
isotype control yourself?) then again, you have different
In the case o
variables. An isotype with a higher F/P than your test
antibody, even if used at the same concentration, will give you
higher "background" fluorescence. (3) Sequence-specific
"nonspecific" binding. Of course the isotype has a different
peptide sequence than the test antibody... are you really sure
that none of these amino acid differences don't contribute to
some selective binding? Until you prove that your isotype
control has the same F/P ratio, and that you are using it at
exactly the same concentrations as each of your test antibodies
(that's a lot of isotype control stains!), then your "control"
is no more than another test sample. That's the kind of
scientific evidence you need to provide before you can use an
isotype to determine positivity in your sample. Isotype
staining certainly has its place. It can indicate IF there is
an issue with background binding. It can let you know that
perhaps you should be careful about interpreting your staining.
And this is particularly true for myeloid cells that have high
levels of FcR. But the problem is that most people go beyond
this "canary in the cave" use for isotypes, and use the isotypes
to set boundaries for gating and identification of positive vs.
negative. And that is where the isotype ceases to be science.
You are correct that we are not in the business of making things
easy. And this is the insidious nature of isotype controls.
They let people think that they are doing something "easily",
when in fact they represent only a crutch that is being
improperly used. They lull researchers into thinking that they
can now identify positive vs. negative. It's so comforting to
think that you have an appropriate control for your staining...
whereas in fact it is much more difficult to properly control
background staining. Finally, I want to address your statement:
"...no scientific evidence..." When I hear of people who titrate
their isotype control to give lower levels of background (to the
same level as their positive antibody).... well, I don't need
"scientific" evidence. Legally, this could be referred to as
"prima facie" evidence of bad science. You don't need
scientific evidence to prove an artefact you need scientific
evidence to prove positive results. The statement that there's
no evidence that the use of isotype controls has hampered any
results is very much like the current "arguments" made by the
religious conservatives in the US in favor of "intelligent
design" (vs. evolution). There we go: experiments using
isotype controls to define gating boundaries... are the
"intelligent design" of experimental analyses
mario roederer, stanford
blocking
I have not had any problems (background staining) with mouse
MEFs so far. For routine Fc blocking when using mouse tissue
samples, I incubate the cells with 2.4G2 (anti-Fc) for 20-30
minutes on ice. Usually, I do not even wash the cells after
blocking (a quick spin for removing 2.4G2 supernatant). It
works really well for me. Occasionally, in my liver MNC
preparations, I do get background B cell staining in spite of
2.4G2 block.
I have not got around this problem yet (other
than using a negative B220 gate).
I have not used the Fc block after the first blocking step. I
block the cells first with the anti-Fc antibody, surface stain
and directly fix and permeabilize my cells for intracellular
staining. Hope this helps.
Sriram
Venkataraman Sriram, PhD
The Walther Cancer Institute
Indiana University School of Medicine
Indianapolis, IN 46202
2.
In our hands we use a routine staining buffer (PBS) that
includes Fc block in all staining and washing steps. I use 2.5%
total protein in the block, 1% BSA,1% FBS and .5% nmIg. It can
be pricey if you use at the recommended concentrations we are
used to so we grow it up with a hybridoma Ig spitting cell line
from ATCC ; MOPC-31C for staining mouse lymphocytes at 500ug/ml.
The Bible for my generation was and is Harlow and Lane,
Antibodies-A Laboratory Manual, Cold Spring Harbor Press for all
this kind of stuff..
cell cycle/subg1 etc -apoptosis
Geert Martens wrote: >we have done a series of experiments with
mitochondrial poisons (such as >rotenone) in rat pancreatic beta
cells, and tried to determine mitochondrial membrane potential
on a semi-quantitative basis with the dye JC-1 we believe our
system works fine : CCCP 10 µM for 20 minutes decreases red
>fluorescence en shifts all cells to green fluorescence, and we
are able to produce classical JC-1 dot plots (FL2 vs FL1 ) were
uncoupling shifts the cells from upper left quadrant (high red,
low green) to lower right quadrant >(low red, high green
fluorescence) however, we would like to know how to convert
these dot plots in CORRECT >numerical data. in If a ratio of
parameters is useful, the thing to do is get a value of the
ratio for each cell and then plot the distribution of values of
the ratio. It is often necessary to scale the ratio values so
they fit on the same measurement scale as the original
parameters. When taking a fluorescence ratio, you need to use
linear values of the data points, not log values; to scale the
values of the ratio, you must multiply the raw values by a
constant. If your data are on a log scale, you can obtain the
log of the ratio a/b by subtracting log b from log a; to scale
this value, you add a constant rather than multiplying, because
the log of a product is the sum of the logs of the multiplier
and multiplicand. However, the arithmetic is only worth the
effort if the two parameters used in the ratio are very well
correlated with one another, i.e., if they form a "long, skinny
cluster" which comes up in different regions of a 2-D
measurement space (e.g., a dot plot) under different
experimental circumstances. If the two parameters aren't well
correlated, the distributions of the ratios won't discriminate
much better between cells in different states than will the
original parameter values. If the parameters are well
correlated, you need to construct a calibration curve relating
the scaled ratio values to what you are trying to quantify, in
this case, mitochondrial membrane potential, meaning that you
have to have some way of setting that to known values. A good
illustration of this methodology, dealing with bacterial
membrane potential measurement using DiOC2(3), appears in Novo
D, Perlmutter NG, Hunt RH, Shapiro HM: Accurate flow cytometric
membrane potential measurement in bacteria using
diethyloxacarbocyanine and a ratiometric technique. Cytometry
35:55-63, 1999; the material is also presented on pp. 256 and
400-402 and on the back cover of the 4th Edition of Practical
Flow Cytometry. Typical clusters representing JC-1 red vs.
green fluorescence (presumably mitochondrial) under different
experimental conditions are not nearly as well correlated as
clusters representing green vs. red DiOC2(3) fluorescence in
bacteria (in the 4th Edition, compare Figure 7-31, p. 399, and
Figure 7-32, p.400). It thus seems doubtful to me that there is
much reward to be gained from going to the trouble of
calculating, scaling, and plotting fluorescence ratios. -
Howard M Shapiro
Histone H3 is phosphorylated at Ser-10 during mitosis and there
is an antibody that specifically detects the phosphorylated
epitope of histone H3 (e.g. provided by Sigma Chemical Co). In
our hands this Ab was the most reliable marker of mitotic cells
(identified from prophase to telophase) applicable to cytometry
(Juan et al., Cytometry 32:71-77;1998). Phosphorylated histone
H3 and other markers of mitotic cells are reviewed by Juan et
al. (Methods to identify mitotic cells by flow cytometry. Meth
Cell Biol, 63: 343-354, 2001)
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
Jake Jacobberger is correct. Most likely the phenomenon reflects
rapid diffusion of the dye and/or ions from the core sample
stream to the sheath stream when they meet upstream in the flow
channel. The diffusion leads to a decrease of dye concentration
in the sample (core) stream which breaks the equilibrium between
the dye and its binding sites in the cell. The changeable
staining pattern is observed until new equilibrium establishes
which takes some time of flow run. We observed this phenomenon
using acridine orange, the dye that is extremely sensitive with
respect to even minute change in its concentration or
concentration of counterions such as sodium or divalent ions in
the sample stream. The phenomenon is additionally exacerbated in
instruments that have long sample lines such as old Ortho
instruments and can be diminished by faster flow rate. We
underscored this in our old papers describing the use of
acridine orange (e.g. Darzynkiewicz, Z.: Simultaneous Analysis
of Cellular RNA and DNA Content. In: Methods in Cell Biology,
Flow Cytometry (2nd edition). Z. Darzynkiewicz, J.P. Robinson
and H.A. Crissman (eds.), Academic Press, New York, N.Y. 1994,
pp. 401-420, see pages 411-412.) I wish Merry Christmas, Happy
Holidays, and the very best in the New Year to all FLOWERS,
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
Hello Janet, Late apoptotic cells have many features similar to
these of necrotic cells, the most apparent one is loss of plasma
membrane integrity. Because they do not exclude 7-AAD, PI or
DAPI the dye exclusion marker is not much help to distinguish
them from necrotic cells. Microscopic examination is obviously
the gold standard to distinguish apoptosis from necrosis so if
you cytospin the cells from parallel sample that was measured
and see only apoptotic cells then you may define that the cells
in the far-left peak are indeed late apoptotic. If not, I would
suggest that you use another marker, such as PARP cleavage or
caspase-3 activation (each of them can be detected
immunocytochemically) as a marker identifying apoptotic cells.
Different strategies to distinguish apoptosis from necrosis are
presented in our chapter: Darzynkiewicz Z, Bedner E, Traganos
F. Difficulties and pitfalls in analysis of apoptosis. In:
Methods in Cell Biology. Vol. 63, CYTOMETRY, 3rd Edition.
Z.Darzynkiewicz, J.P.Robinson, and H.A.Crissman, Eds. Academic
Press, San Diego, CA, 2001; 527-559
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
The Chromatin Structure Assay is in reality analysis of
susceptibility of DNA in situ to denaturation, induced by acid
or heat. Extensive studies have been carried out using this
assay to analyze chromatin of different cells in relation to the
cycle phase, three decades ago. For example, when applied to
lymphocytes this assay allows one to discriminate Go from G1
cells and G2 from mitotic cells, as well as distinguish other
phases of the cell cycle. More recently this assay was
mentioned, with other "historical" methods applicable to cell
cycle, in the review article in Cytometry (Cytometry of the cell
cycle. Cycling through history. Cytometry, 58A; 21-32, 2004).
The original, earlier papers on this topic are: (1)
Darzynkiewicz, Z., Traganos, F., Andreeff, M., Sharpless, T.,
Melamed, M.R.: Different sensitivity of chromatin to acid
denaturation in quiescent and cycling cells as revealed by flow
cytometry. J. Histochem. Cytochem., 27:478-485, 1979;. (2)
Darzynkiewicz Z., Traganos, F., Sharpless, T., Melamed, M.R.:
Cell cycle related changes in nuclear chromatin of stimulated
lymphocytes as measured by flow cytometry. Cancer Res.. 37:46354640, 1977. The confocal analysis of DNA denaturation by this
assay is described in: Dobrucki J.,Darzynkiewicz, Z. Chromatin
condensation and sensitivity of DNA in situ to denaturation
during cell cycle and apoptosis. A confocal microscopy study.
Micron, 32: 645-652, 2001
.
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
Dr. Salinas inquires about caspase-8 assay by flow cytometry.
The most specific approach would be to use Ab that reacts with
the activated form of caspase-8 but not with pro-caspase-8.
Although large number of caspase-8 Ab are commercially
available, I have not seen yet the published data showing the
use of Ab to detect activation of caspase-8 by flow cytometry.
Abs against activated caspase-3 and -9 are listed in some
catalogues
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
Fluorescent tagging of DNA can be accomplished by the
photolabeling technique, utilizing photoactivated ethidium
monoazide (e.g. Riedy et al., Cytometry, 12: 133, 1991).
Zbigniew Darzynkiewicz
The method to differentially identify DNA replicating and
apoptotic cells is described by Li et al., in Experimental Cell
Res. 222, 228-237,1996. In the first step the existing DNA
strand breaks in apoptotic cells are labeled with fluorochrome
of a particular color using exogenous terminal transferase
(TUNEL). Subsequently, the cells are illuminated with UV light
to pholytically induce DNA strand breaks at the sites of BrdU
incorporation. These, in turn, are labeled with another color
fluorochrome using the same principle of labeling (TUNEL). DNA
can be then counterstained with still another color dye to
obtain simultaneous differential staining of apoptotic- vs. BrdU
incoroporating- cells, and discriminate G1 vs. S vs. G2+M cells
in both, apoptotic and nonapoptotic populations
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
To the ongoing discussion about obtaining cells synchronized in
the cycle I would like to add the following warning: The
synchronization by transient cell arrest in the cycle induces
growth imbalance and dramatically alters expression of cyclins
and other cell cycle regulatory proteins. For example after
double thymidine block we have observed "unscheduled" expression
of cyclins B1 and A in cells at the G1/S boundary, over fivefold increase in expression of cyclin E, and 40% increased total
protein content [Gong et al., Cell Growth & Differentiation, 6:
(November issue) 1485-93, 1995]. Kinetic and metabolic
properties of so synchronized cells are much different compared
to the cells from asynchronous, exponentially growing cultures.
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
The problems and difficulties in analysis of apoptosis,
particularly of adherent cells, we reviewed in Meth Cell Biol
63: 527-548,2001. Extensive trypsinization and repeated
centrifugations may indeed affect Annexin V assay. In our hands
the immunocytochemical detection of cleaved PARP (p89) (Exp.
Cell Res., 257: 290-297, 200) or of activated caspase-3
(Cytometry, 55A, 50-60, 2003) appear to be both highly sensitive
and specific markers for adherent cells. Also sensitive is
detection of caspase activation by FLICA.
One has to be
concerned, however, that during apoptosis the cells detach
themselves and float in the medium. Assessing apoptotic index in
cultures of adherent cells, thus, to account for the floating
apoptotic cells, one has to collect the medium, centrifuge it
and pool the floating cells with the trypsinized cells.
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
Cyclin D1 can easily be detected - one can follow the protocols
in Current Protocols in Cytometry, section 7.9, (see also
Cytometry, 25: 1-13, 1996). However, the "scheduled" expression
of cyclin D1 is rarely observed, as most tumor cell lines are
very variable and may also express it in all phases of the cell
cycle.Likewise, tumor and leukemic cells from patients. We have
seen, however, that when normal cells, (e.g. fibroblasts) are at
perfectly exponential growth phase (low cell density, 2 - 3 days
after re-seeding) they invariably express cyclin D1 only in
early portion of G1, and perhaps very late in G2 (few cells in
G2/M peak); The cells in S and most G2 cells were totally
cyclin D1 negative. Medium change, prior trypsinization (<12 h)
or sub-confluency dramatically altered expression of cyclin D1
even in normal cells, which showed then "unscheduled" pattern of
its expression vis-a-vis the cell cycle phase. We have also
noticed that cold methanol cell fixation and Ab then from
Immunotech (Now Coulter-Immunotech) were superior than ethanol
fixation and other Abs
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
Let me add a comment regarding cell killing by pyronin Y. Unlike
in fixed cells, where pyronin Y binds to RNA, in live cells (at
low concentration) it accumulates quite selectively in
mitochondria. This makes the cells photosensitive. A short
exposure to light disrupts mitochondria and rapidly kills the
cells (e.g. "Cytostatic and cytotoxic properties of pyronin Y:
relation to mitochondrial localization of the dye and its
interaction with RNA", Cancer Res., 46: 5760-5766, 1986). In the
dark, however, at 1.7 - 3.3 uM concentration, it is cytostatic
(G1 arrest) when continuously present in the culture. At higher
concentration (>6 uM) it seems to bind also to nucleolar and
cytoplasmic RNA and arrests cells in S and G2/M.
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
The cells that are advanced in process of apoptosis lose DNA by
shedding apoptotic bodies that contain granules of chromatin.
Furthermore, when DNA fragmentation by CAD (caspase activated
DNase) is extensive, DNA fragments become small (size of mononucleosomal DNA), and such fragments may not be properly fixed
(crosslinked to protein) with formaldehyde -they may leak out of
the cell during the fixation and staining procedure. Thus, the
late apoptotic cells, even after fixation with formaldehyde (as
it is in the case of ApoBrdU assay), may have a deficit in
(fractional) DNA content. With a gross loss of DNA, fewer DNA
breaks (3' OH termini) remain in the cells to be labeled with
BrdU. Hence, these late apoptotic cells may also show a
decreased BrdU-associated fluorescence.
I would suggest
to classify the events with DNA content lesser than 10% of the
mean DNA content of the G1 cells as apoptotic bodies, while the
events with DNA content (DNA-associated fluorescence) between 10
% to up to G1-cell cluster peak, as apoptoptic cells. Although
some of the events with less than 10 % DNA than G1 cells may in
fact very advanced in apoptosis cells, most such events are
expected to be apoptotic bodies or cell fragments. It seems,
therefore, that it is a lesser error to classify them as
apoptotic bodies or fragments of apoptotic cells, than as
apoptotic cells.
Unfortunately intensity of light scatter
signals (FS vs SS) is not much of help, because the very late
apoptotic cells and large apoptotic bodies or cell fragments may
have similar light scattering properties. The "veteran"
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
Repeated centrifugations in general can make non-apoptotic cells
annexin V-positive. Furthermore, monocytes phagocytize
apoptotic bodies that are shed from the neighboring apoptotic
cell, and in the process become "false-positive" apoptotic
cells, by the annexin V assay (e.g. Marguet et al., Nature Cell
Biol., 1999;1:454-56). This is likely to be due to the fusion of
plasma membrane of apoptotic bodies with membrane of monocytes.
It has to be stressed that density gradient separation of cells
to estimate apoptotic index may introduce additional bias
because nucleus and cytoplasm undergo condensation during
apoptosis. Hence, density of apoptotic cells is markedly
increased, and they may be lost from the gradient, where one
expects to find them if they would be non-apoptotic (e.g.
mononuclear cells band on Ficoll-Hypaque gradient). These and
other potential traps and difficulties in estimation of
apoptotic index are discussed at length in Vol. 63 Methods Cell
Biology/Cytometry, (2001;63: 527- 546).
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
The most common cause of the "sub-G1" spreading to very low DNA
values is inapppropriate cell preparation. The cells have to be
fixed in the preciptating fixatives (e.g. 70 % ethanol) then
hydrated, stained with PI or DAPI and measured. The fragmented,
low MW DNA undergoes extraction from apoptotic cells upon their
hydration and apoptotic cells usually end up with about 20 - 50
% DNA of that of G1 cells, forming a distinct sub-G1 peak. If
necessary, one can enhance the extraction using high molarity
phosphate buffer. It is a common practice, however, to lyse the
cells in hypotonic buffers or buffers that contain detergents.
When a single apoptotic cell is lysed it can release many
chromatin fragments. Because these fragments have minimal DNA
content logarithmic scale is then used, and the fragments are
erronously identified as individual apoptotic cells. Obviously,
under these conditions a single apoptotic cell may generate up
to a dozen, occasionally more object counted as "apoptotic
cells" . Needless to say, individual chromosomes from mitotic
cells, micronuclei, etc, are also misclassified as apoptotic
cells.The light scatter signal from lysed cells is not much
informative. It should be noted that on rare occasions apoptosis
may proceed very rapidly and DNA degraded extensively that even
after fixation and apoptotic cells may end up with less than 20%
DNA of that of nonapoptotic cells. In such an instance one may
fix cells briefly in formaldehyde (1%, 10 min), to prevent
leakage of the low MW DNA, and follow by fixation in 70%
ethanol. These problems and potential pitfalls are discussed in
Meth Cell Biol Vol 63, pp 257-546, 2001
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
The "best" positive control: HL-60 cells treated with >0.15 uM
camptothecin (CPT) provide a reliable model of apoptosis. Most
apoptotic changes (mitochondrial, plasma membrane, DNA
fragmentation, nuclear fragmentation) occur during the initial 4
h of the treatment. The advantage of this model is that only S
phase cells undergo apoptosis (Del Bino et al., Cancer Res., 51:
1165, 1991). Thus, G1 and G2/M cells, within the same sample may
serve as a negative control. The critical point is the cells
have to rapidly progress through S phase to be sensitive to CPT.
It is a collision between the progressing DNA replication fork
and the lesion iduced by CPT that provides the signal inducing
apoptosis. Any slowdown in S phase progression, therefore, such
as due to higher density of cells in the culture
(subconfluency; > 800.000 cells per ml) makes them less
sensitive to CPT.
2. The issue as to whether the second "p" is
silent is a subject of long and ongoing dispute. Interestingly,
it become apparent quite recently that the term was already used
by the father of medicine Hippocrates, to describe the falling
of the bone fragments during healing , i.e. in the context
related to its common use (see Esposti: Cell Death & Differ.; 5:
719, 1998). As a Greek word, it should be pronouced with two "p"
(see Funder, Nature, vol . 371, 1994 (Sept. 8 issue)"Apoptosis:
two p or not two p". English authors, however, often transform
its prononciation to English language suppressing the second "p
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
It is difficult to answer to that question because the
distinction between "Go" or "completely out of the cycle" or
even "slow mode G1" is, to some extent, in semantics. The
concept of Go cells was introduced by Lajtha four decades ago
[Lajtha LG, Haemopoietic stem cells: concept and definitions,
Blood Cells, 1979; 5: 447-55] as an operational term, to define
the cells which do not enter S phase (incorporate 3H-thymidine)
for the duration of at least two cell cycles. While the
hematopoietic stem cells or peripheral blood lymphocytes are
considered to be an example of Go cells, this term has been
used by many authors indiscriminately to describe noncycling
cells in general, in a variety of cell systems, including
cancer cells. The first (metabolic) marker shown to distinguish
Go from G1 cells was cellular RNA content reflecting the number
of ribosomes per cell. Go cells, contain on average 10 times
fewer ribosomes compared to cycling G1 cells (e.g. Stanners et
al., J. Cell Physiol, 11: 127, 1979) and can be distinguished
from G1 cells by flow cytometry based on their minimal RNA
content (PNAS 73: 2881-6, 1976). Another metabolic attribute
(the one that can be used supravitally) that distingushes Go
from G1 cells, is the very low uptake of mitochondrial probe
rhodamine 123 by the former (PNAS; 2383-2387, 1981). We reviewed
all the differences in metabolic parameters between cycling and
noncycling, "genuine Go" and "slow G1", or "quiescent" cells,
and this allowed us to subdivide the cell cycle on several
subcompartments, that can be identified by cytometry (Cytometry,
1:98-108, 1980). Since then the Ki67, cyclin D, cyclin E and
status of phosphorylation of pRB were proposed as new markers
distinguishing Go from G1 cells. Based on differences in
expression of these proteins and of pRB phosphorylation we
proposed subdivision of the Go-G1 phase on additional
subcompartments (Cytometry, 25: 1-13, 1996). It should be
noted, however, that cells of most tumors and transformed cell
lines are unable to enter the state that would be characterized
by RNA content or by cyclin expression as Go. They often express
cyclins in "unscheduled" way, e.g. presenting the G2 cyclins A
and B1 in G1 phase. In conclusion, in the unclear situation,
instead of classifying cells as "Go" or "noncycling G1" etc, I
would characterize them based on the measured molecular or
cytometric attribute(s) such as "cyclin D-negative" or "cyclin
D-positive a and cyclin E negative", etc
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
There are numerous methods that allow one to define in which
cell cycle phase cells are dying by apoptosis. The most widely
used is TUNEL assay. We reviewed these methods many times, -
e.g. in Methods in Cell Biology, Vol 75 (2004) Chapter 12:
"Cytometric Methods to Detect Apoptosis". It is even possible to
define whether G0 or G1 lymphocytes undergo apoptosis by
measurement of cellular DNA and RNA content after staining cells
with acridine orange (e.g. see Huang et. al. Cytometric
assessment of DNA damage in relation to cell cycle phase and
apoptosis. Cell Proliferation, 38: 223-243, 2005
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
While fixation in formaldehyde is required to detect apoptotic
cells in the TUNEL assay, formaldehyde should not be used in the
assay based on analysis of cellular DNA content ("sub-G1" cell
population); ethanol at 50 - 80% concentration is preferred.
Unlike formaldehyde, fixation in ethanol does not crosslink DNA
and thus allows the low molecular weight fragmented DNA to be
extracted from the cells when they are transferred from ethanol
to buffer or PBS, incubated with RNase and stained with PI or
DAPI, so apoptotic cells may end-up with fractional DNA content.
In fact, when cells are fixed in formaldehyde, apoptotic cells
often cannot be identified as the sub-G1 population. It is only
when the apoptotic process is very advanced and some DNA is
being lost by shedding apoptotic bodies that contain parts of
fragmented nuclei, apoptotic cells may have fractional DNA
content and be distinguished as "sub-G1" cells after
formaldehyde fixation. It is also worth to notice that if G2M
cells undergo apoptosis they may end-up with a "sub-G2M" DNA
content which may locate them at the site of S-phase cells on
the DNA content histograms
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
Apoptotic cells are strongly labeled in TUNEL assay only when
they have a large number of DNA strand breaks. This is the case
when DNA fragmentation is very extensive, which occurs when
internucleosomal DNA sections are cleaved. However, in some
cell types (often of epithelial or fibroblast lineage) or
instances DNA fragmentation stops at the initial step i.e.
generating 300 - 50 kb DNA sections, and does not progress into
intenucleosomal (~180 bp) sections. In the TUNEL assay such
cells are only weakly labeled. It should be noted that with most
TUNEL kits (including APO-BRDU) a positive control cells are
provided. They are camptothecin-treated leukemic cells which are
expected to have internucleosomal DNA cleavage in about 30-40 %
of the cell population (S phase). This control, if found to be
TUNEL positive, provides an assurance that the kit is OK.
Furthermore, in the case when the control is positive but the
investigated apoptotic cells are negative, it provides evidence
that DNA fragmentation in the studied cells did not progress
into internucleosomal DNA sections.
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
Excellent method to stain DNA in yeasts for cell cycle analysis
is described by Steven Reed in CELL CYCLE. Below I am providing
the link to CELL CYCLE (which still is available online). His
atricle in in issue # 2. Dr. Reed also prepared a protocol on
this method which is being now included in Current Protocols in
Cytometry.
Zbigniew Darzynkiewicz, M.D., Ph.D.
Research Institute
Brander Cancer
clumping
* There are several tips for preventing clumping. Using EDTA at
up to 5mM concentrations may help prevent cation dependent cellcell adhesion. Do not keep the cells at too high a concentration
either (no more than 5-6 million/ml). Likewise using Ca/Mg++
free buffers will also help. Remember adding serum to your
buffers will replace the Ca/Mg++ so if you need a
protein support try using BSA instead. If your cells are
clumping due to dying cells, the addition of DNAse II (10u/ml)
may help. Of course you could filter your sample through a nylon
mesh but the important thing is to
prevent clumping so you do not lose too many cells.
* I use 2-5% BSA to coat the cells. I think it also helps
protect the cells,
when sorting, from the degassing effects at the
nozzle exit. If they are really clumpy I add some DNAse
[assuming the cells are alive].
data presentation
There's been extensive rigorous analysis of %Positive
quantitation with dim populations--look it up in the
literature, there are a number of papers (see bottom of email).
Many third party software programs support these types of
analyses; for example, FlowJo can compare the negative and
stained controls using the five somewhat-related algorithms
devised by Roy Overton, Bruce Bagewell ("SED"), Cox (Cox chisquare), Kolmogorov and Smirnoff ("K-S"), as well as by our own
group ("PB" or probability binning). In general, these
algorithms agree quite well--although the Cox, KS and PB
methods aren't strictly %Positive quantifiers (but can be
adapted to be such). In my opinion, for %Positive, the best is
the SED algorithm. This has significant support both from
basic mathematical principles as well as from empirical
analyses. The PB method has the unique facet that it can be
used to "gate" on cells that are different (e.g., positive) in
one or more dimensions. Now let's dispense with a few myths
that you brought up! >... When the whole peak shifts, the whole
population is brighter >than the Negative control population.
That means it's 100% positive >- including those dim cells in
the 'positive' peak that aren't as >bright as the bright cells
in the negative peak. That's not really true, for a number of
reasons. First, you haven't defined what you mean by "peak".
If you mean the "mode" (which is what most people mean when
they think of peaks), then it's not at all true; the mode can
be significantly influenced by changes in underlying
representations of positive and negative; an increase in the
mode does not mean that the cells are 100% positive. Even if
the "whole peak" (by which I assume you mean the bottom
percentile as well as the top percentile) moves, this does not
indicate that all the cells are positive! Consider the simple
example of an unstained population that is actually comprised
of two sets of cells: A & B, where "B" cells have slightly more
autofluorescence than "A", but the mixture doesn't resolve and
appears to be a single peak. After staining, all of the "A"
cells become slightly positive, and are slightly brighter than
the B cells, which are still negative. Again, the distribution
doesn't resolve into 2 peaks. That's a simple case where half
of the population is staining, yet "the whole peak shifts"! Is
this a trivial example? Certainly not: Within lymphocytes, B
cells and T cells have different autofluorescence levels--so if
you were to stain one population only with a dim reagent, you
might mistakenly conclude that all of the lymphocytes express
that antigen! >Back to the 'small differences' case: If your
negative control is >in one location, and the negative cells in
the test sample are in >the same location but there are a few
bright cells, then you can use >frequency analysis to get the
percentage of those positive cells >(use a 2-parameter plot and
a polygon region - NEVER a histogram). Well, that's a blanket
statement that I must also disagree with! Why "NEVER" use a
histogram? Admittedly, I use bivariate plots often to gate
essentially one a one dimensional expression. But I do so with
guilty pleasure. The claim appears to be that you can better
separate the dim positives from the negatives on a bivariate
display. And this is visually supported in many cases.
However, this is purely a visual artifact! It's magic!
It's not mathematically true! In fact it's.... myth!
EXCEPT when there is a relationship between the expression of
the dim marker and the measurement on the other axis (and there
often is--particularly with something like SS or FS, when there
is a size-dependence). If that's the case, then you can't use
just any bivariate display, you must use the bivariate display
of your interesting marker against the parameter which provides
additional information. If the other parameter in the
bivariate display is not mathematically related to the
measurement marker, then there is no scientific basis for
stating that the resolution of the dim cells (ability to gate)
is better in a bivariate display.
And yes, I'd be happy to
follow this up with real math if necessary. One thing to
consider is that dot plots are heavily influenced by the number
of events you collect. Pretend that you had collected a
trillion events instead of 10,000 -- all of a sudden, the
distinction on that bivariate plot has disappeared! (And yet,
the histogram looks no different). Furthermore, the assertion
that gating on bivariate plots is better than on histograms
belies the underlying assumption that the gating is completely
subjective! Don't be misled by the typical elliptical (or
circular) distribution of events in a bivariate display of
uncorrelated parameters -- this does not help you identify
boundaries any better than from a histogram, except in a
subjective manner. Of course, there's nothing wrong with
subjectively placing gates, as long as you are aware that this
is the case. But if your are concerned about accurately
estimating %Positive, then certainly any subjectivity in gate
placement must be removed. Incidentally, the algorithms
referenced above do an excellent job of estimating %Positive,
whether the expression is bright OR dim. Manual gating fails
miserably if there's no defined separation. >
If you have
brighter events AND your negative peak moves up, you >either
have 100% positivity in your sample (with 'dims' and
>'brights') OR your negative control isn't working properly and
you >only have a few bright positive events.
OR... your
negative control works just fine, it's just that the stain has
some nonspecific binding on the nonexpressing cells! Oh wait -this means your negative control isn't an adequate control...
but then, that's almost always true. It's nearly impossible to
have the perfect negative control. (And please, don't even get
me started on isotype "controls" -- something I want to rename
as "isotype uncontrols"). Nonetheless, the point is that there
are lots more possibilities than the two you mention. >PS-The
training videos will be available in October. Well, great! ...
but I hope they carry a bit more rigorous explanations than
your original response... Perhaps the self-assignment of the
moniker "FlowJock" is a bit premature. (PS, I sincerely hope
you don't try to claim a trademark on a term that has been in
general use by the community for many years--that would be a
waste of effort and community good will). mr (you may consider
me as an untrademarked FlowJock)
1) Overton WR. Modified
histogram subtraction technique for analysis of flow cytometry
data. Cytometry. 1988 Nov;9(6):619-26.
3) Roederer M,
Treister A, Moore W, Herzenberg LA. Probability binning
comparison: A metric for quantitating univariate distribution
differences. Cytometry. 2001 Sep 1;45(1):37-46.
4) Roederer
M, Moore W, Treister A, Hardy RR, Herzenberg LA. Probability
binning comparison: a metric for quantitating multivariate
distribution differences. Cytometry. 2001 Sep 1;45(1):47-55.
5) Roederer M, Hardy RR. Frequency difference gating: A
multivariate method for identifying subsets that differ between
samples. Cytometry. 2001 Sep 1;45(1):56-64. 6) Cox C, Reeder
JE, Robinson RD, Suppes SB, Wheeless LL. Comparison of
frequency distributions in flow cytometry. Cytometry. 1988
Jul;9(4):291-8.
Received on Wed Sep 15 15:06:28 2004
mario roederer, stanford
I liked Ray's and Mario's approach to show a few examples. It
shows the artistic freedom one can get on how to display the
data. And they only used one data set and two software packages
and far from every possible iteration of scaling smoothing and
colour. And they happily accepted Fluor:CD8 as an axis label. I
would have thought it to be fluorescent if that is what Fluor
means. I tend to write for example CD8*FITC 525nm log which
still doesn't give you bandwidth etc but tells you at least it
is green fluorescence. I actually love density plots (please
don't call them colour dot plots as this tends to be used for
colour gated dot plots) over contours and actually most of all a
3-D display where I can see mountains or clouds. Unfortunately
we can not yet print the moving pictures described in Harry
Potter but that is when the fun really starts. However, it can
be done in on-line publications. The confusion created by our
beloved contour or dot plots can be enormous. Not for no reason
one of Marc Abraham's AIR-logo's shows a dot plot under the
stinker (http://www.improbable.com/bookstore/bookstoretop.html). Actually nicely shown in Mario's example of smoothed
contour plots is how you can make cells vanish. Are these small
populations between the single and double positive cells
important? or perhaps a compensation error?... If one wants to
show those cells to make a point, one would not use the smoothed
contour plot. If one was to sort them, the contours would lead
nowhere. From that point the old Ortho 50H software with it's
grey level dot plots was far advanced at it's time. With 16 grey
levels it allowed a huge dynamic range for density plots (e.g.
1=2^0 to 32768=2^15 per channel) w/o compromising the
resolution at the bottom - unless you do not like the log-2 z-
scale. Let's step back a bit Why do we publish? Normally to
convince someone else about the importance and values of our
work. Why would we add those funny little pictures? To convince
the reader with a visual aid about our data, the population we
have seen doing something, e.g. once you seen a cluster - you
shall believe. They are descriptive sketches and nothing else
as they don't give is any statistics themselves. In particular
with low event numbers it becomes very important to reflect on
confidence intervals of your data as nicely presented by Terry
Hoy on the Royal Microscopical Society Flow Course is Sheffield
a couple of weeks ago. However, the plots have something in
common with statistic: Statistics are like a bikini - what they
reveal is suggestive, but what they conceal is vital. (Aaron
Levenstein)
Now if we argue the use of plots for illustration
only but the use of "solid numbers" to substantiate our work
with "sound statistics" we have to keep in our mind that all
this number crunching only characterises / validate the position
of the dots on the screen, nothing else. We can only test the
numerics of our measurements and not the measurement itself.
Apart from the requirement for correct instrument set-up this is
subject to scientific reasoning; about how the measurement was
performed and what controls were done, if saturation was reached
etc. in order to interpret the indirect measures we have
undertaken. This includes detailed description of clones and
concentrations and control data and "unmassaged" pictures of raw
data and gated data. The question remaining is how to validate
the quality of the data and who is to do it. For validation in
house for example we display / print all fluorescence channels
versus log side scatter ungated. For whole blood this helps as
it gives you already a differentiation of lymph's, mono's and
neutrophils (and eosinophils if for example using FACSlyse).
Thus if you suddenly spot CD8 positive neutrophils you know that
there is trouble. For cultured cell lines log forward scatter
vs. fluorescence seems to be better as in a lot of cases the
cells with lower forward scatter are the ones on their way out.
This type of display also indicates the presence of fluorescent
aggregates that can lead to problems by sticking to or
coinciding with cells (how do we want to report coincidence
factors?) and shows if the antigen expression is scatter /
volume related or not. But this is all a matter of personal
taste. I do not believe we can eradicate poor publications. On
one side there is always human error. On the other side there is
an incredible inflation of science and in particular the numbers
of publications and journals, inevitably leading to a decay in
quality. We can only try to educate people in their ability to
set up experiments and analyse data. Attempts like Mike's CD-ROM
approach or Paul's virtual laboratory where you can even do the
virtual pipetting of antibody are leading in the right
direction. The latter could even be spiked with pitfalls like
changing the buffer batch in an experiment, picking one made up
with water contaminated with red fluorescent algae. Now in a
proper clinical setting, as I know from my brother, all the
results have to be signed off and interpreted by him or a
colleague as a specialist before they leave the lab for the
physicians out in the University. Unless you run your samples in
a core facility with an experienced cytometrist, such gate
keepers may not exist. For political financial and logistic
reasons a lot of people have bought their own instruments
independent from an already existing expertise. The time and
effort put into the proper planning and analysis of experiments
by someone who knows his cytometry would cost (not money
necessarily). If the gate keeper is to be the reviewer of an
article then he should not only be skilled enough to understand
the flow data but he also would require some validation
information even if not published as it is not possible to judge
complex work from one or two pictures. In that context I think
that the suggested discussion board for post publishing review
offered via the publisher to their subscribers speaks in favour
of the publisher, as this type of feedback does allow him to
improve the journal quality. I am sure we can come up with a
suggested list of information that should be included on
cytometry experiments if applicable and make it easier not to
forget the necessary controls and I would ask James Watson
(http://www.cyto.purdue.edu/flowcyt/books/bookl.htm) to join the
advisory party that might be set up for that. But there will
always be a lot of poor data published, for numerous reasons,
and the only one to blame is the reader that believes
information to be true because it was printed in a newspaper or
a publication to be correct just because it was published in a
scientific journal (see www.improbable.com for further enjoyment
on that).
gerhard nebe-von caron, unipath
fixation
Paraformaldehyde is a polymerized form formaldehyde. It is
hardly soluble and it cannot be used as a fixative. Only
formaldehyde is used as a fixative. Howeve, formaldehyde in
aqueous solutions spontaneously polymerizes. Therefore, methanol
is often added to slowdown the polymerization reaction.
Solutions of formaldehyde (usually ~ 37%) in water, containig
10-15 % methanol as a preservative are generally called
"formaldehyde"; such solutions are being sold by most reagent
companies. Solutions further diluted (4-10 %) received name
"formalin". Methanol-free formaldehyde, which sometimes is
preferred (e.g. for fixing cells for some some histochemical
reactions or in immunocytochemistry), can be obtained by
hydrolysis of paraformaldehyde. This is usually done by
extensive heating of paraformaldehyde solutions. Because of this
procedure the methanol-free formaldehyde received (incorrrectly)
the name "paraformaldehyde". In the past, this was the most
common way to obtain methanol-free formaldehyde. Unfortunately,
this incorrect name is still often used in the literature,
generating the confusion. The methanol-free formaldehyde
solutions can now be purchased. Some are called "ultrapure". We
purchase such solutions (10%) from Polysciences, Inc. (800-5232575); they can be stored at room temperature. I would not
recommend, however, to store them longer than one year, since
formaldehyde in these solutions still has tendency to
polymerize. It should be noted that all formaldehyde solutions
are highly toxic and carcinogenic.
Zbigniew Darzynkiewicz
Catherine,
Autofluorescence is usually higher with older aldehyde
solutions. I would recommend using freshly prepared formaldehyde
(less than one month old). Also, the addition of 0.3% Triton X100 to the antibody incubation
solutions will decrease autofluorescence.
--Randy
Hi Catherine:
Formaldehydes and glutaraldehydes are bad actors when it comes
to fluorescence-based cytologic analysis. Either get away from
fixation with aldehydes or the answer to the problem is aldehyde
blocking. This is done by reducing the -CHO groups to -OH with
sodium borohydride or by usingbland amino groups (glycine,
bovine albumin, skimmed milk). The other option is to freeze
cells. Make cytospins when you need them.
The third option is to live with the autofluorescence. Just
dedicate FITC channel to autofluorescence.
Autofluorescence Eliminator Reagent
http://www.chemicon.com/featured/autofluorescence.asp
http://www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.p
df
Good luck
Padma
----------------------------------------------------------------------------------------Hi Catherine,
One method is to use the violet laser for autofluorescence
correction.
The blue laser excites both autofluorescence and FITC/Alexa 488.
The violet laser excites only the autofluorescence.
Subtracting the violet excited green scan images from the
FITC/Alexa 488
scan images leaves a close approximation to the specific
fluorescence.
Best wishes,
Ed
------------------------------------------------------------------------------Dear Catherine,
This is a common problem in immuno-histology.
Attached is a paper that may help. I have not tried sodium
borohydride in a flow setting yet, but one of the
researchers who use my flow lab is going to give it a go.
Regards
Rob W
Paraformaldehyde is a white solid polymer (HCHO)n of
formaldehyde where n is at least 6. It is used as a
disinfectant, fumigant, and fungicide. It is formaldehyde
(HCHO), that is used as cells/tissues fixative, not
"paraformaldehyde". Because formaldehyde in solution undergoes
oxidation and polymerization, it is common to make fresh
solutions by depolymerizing paraformaldehyde, most frequently by
heating in water solution. Hence, such product is often
(incorrectly) called "paraformaldehyde". The hydrolysis is a
hazardous procedure, because paraformaldehyde/formaldehyde
vapours are strongly irritating and carcinogenic. To make
solutions of formaldehyde more stable methanol is often added,
and this is called "formalin". Formalin 100% is a soluition of
37% by wt of formaldehyde gas in water, with 10-15% methanol to
prevent polymerization. It is also often called "formalin 40",
as it contains about 40g of formadehyde in 100 g of water.
For
most applications that call for "paraformaldehyde" we use the
methanol-free formaldehyde, that is available from Polysciences,
Inc (Warrington, PA). I do not know how they stabilize their
product, but we store it up to a year, as prescribed by the
vendor, and do not see any deterioration of the results.
Zbigniew Darzynkiewicz, M.D., Ph.D. Brander Cancer
Research Institute
intracellular staining
The most simple and in most cases perfect fixation procedure for
intracellular proteins is using cold 70% Ethanol. It works for
most
proteins, you can store your samples in the fridge for a year or
longer
without any changes in antigen distribution, you can do a
simultaneous DNA analysis for cell cycle etc. Due to the
possibility of long time storage you can analyse all the samples
from an experiment at the same time which improves
comparability. If your protein is very small there may be a
problem with loosing it, although most, even small proteins are
precipitated. There have been some cases described in the
literature that the conformation of some antigens may change so
that the antibodies will not recognize them anymore, but these
cases are very rare. The only thing you have to take care of is
the possible formation of aggregates during the fixation
procedure. Here is the protocol I use for intracellular
immunofluorescence:
Centrifuge your cells in a tube with a conical bottom, discard
the
supernatant, resuspend the cells completely in the remaining
drop. Now
comes the tricky part: Put the tube on a vortex machine, keep it
shaking while adding the ethanol dropwise and slowly. This is to
avoid aggregate formation. Use ~1ml ethanol for 5x10e6 cells.
Keep in the fridge until use.
Before staining centrifuge again, discard supernatant, resuspend
cells in remaining droplet. Add 1ml Buffer (0.1%Tris.HCl, 0.1%
Triton X100, 2mM MgCl2 pH7.4) the same way as when you added the
ethanol (dropwise and slowly while vortexing). Wash again with
the same buffer, again taking care to avoid aggregates. Wash
again with trisbuffer including 20% FCS or 2% BSA and incubate
for 30 min to block unspecific binding sites. Wash again
blocking buffer and include your antibodies. After the last
staining step was with buffer w/o FCS or BSA and analyse.
Hope this helps.
With kind regards,
Nicole
I can recommend CALTAG Fix and Perm for intracellular staining
and I
have used this product in both diagnostic and research
laboratories.
The reagents are easy to use and also allow concurrent surface
staining. The FSC/SSC presentation does not alter significantly
compared to other permeabilising reagents.
I have no commercial interest in CALTAG products and I offer
this
information as my opinion only.
Good luck!
Cathy
-----------------------------------------------------------------------------------------------
Elaine,
In my experience you may need to look at different fixatives as
well as
permeabilization agents.
We are looking at human white blood cells and we need both good
intracellular perm as well as good light scatter properties (for
differentiating the WBC subsets). This may not be a concern for
you since you're using a cell line.
If you're looking at something novel intracellularly, use
something known first to validate your fix/perm . For example,
with our
WBC work we look for good myeloperoxidase labeling in
granulocytes to
validate the fix/perm.
I hope this helps.
Peter
Peter Lopez
---------------------------------------------------------------------------------------------------------------------Hi Elaine,
Ethanol is a penetrating fixative and conditions of use
including cell type will govern the distance of effect into a
cell, for most cells it is a total fixation.
For DNA staining with dyes this is fine but for Ab you have to
know they are against the fixed epitope.
For most Ab used in Flow Cytometry it is preferable to Fix the
cell membrane with paraformaldehyde then use a detergent to
permiabilise said memebrane.
FIX
Paraformaldehyde fixative solution PFM
4 % (w/v) paraformaldehyde
3 % (w/v) sucrose ( I never used sucrose in my
fixation mix
only in the Hypertonic solution desined for membrane
shedding to recover nucli. You could try it for
comparison)
in PBS pH 7.4.
Store at 4°C in the dark for a few days.
PERM
Triton permeating solution
1 % (v/v) Triton-X100
in PBS pH 7.4.
Store at room temperature for a few weeks.
Saponine permeating solution
0.1
% w/v
in PBS pH 7.4.
Store at 4°C for a few days.
Nonident permeating solution
0.5 % (v/v) Nonidet P-40
in PBS pH 7.4.
Store at 4°C for few days.
N-OctylGlucosamine (NOG) permeating solution
0.74 mg/l (w/v) NOG (This is the Critical Micelle
Concentration for this detergent)
in PBS pH 7.4.
Store at 4°C for few days.
The problems with " home brew" are reproduction, QC and shelf
life so I went to a commercial product. We sell the IntraPrep
kit for this which has a PFM fixation and Saponin Perm. 50 test
IM2388 and 150 test IM2389 As a positive control to demonstrate
perm and staining we have anti-Tubulin conjugated with FITC part
6607113
Regards
Martin
---------------------------------------------------------------------------------------------Hi Elane,
I did some intracellular staining in thymocytes some time ago. I
fix my
cells with formaldehyde solution.
My experience was that is was critical, how fresh the
formaldehyd solution was. I took 2% formaldehyde and end up with
1% end concentration for fixation.
I attach a protocol in pdf format and hope this helps a bit
Good luck
Steffen
====================
Dr. Steffen Schmitt
--------------------------------------------------------------------------------------------------------Elaine,
We've actually pondered this same question at Cell Signaling
since our
customers use a variety of different fix&perm methods. We have
settled on a protocol that involves fixation in 1% formaldehyde
for 10 min at 37C, then permeabilization in 90% methanol. This
protocol works very well for every antibody we tried. Some of
our collaborators recently compared this protocol to a saponinbased fix&perm kit from Invitrogen and screened with a number of
antibodies. All of the antibodies worked well with the
aldehyde/methanol, but over half of them did not work at all
with the saponin-based kit. The only down-side to methanol
permeabilization is that the scatter characteristics of the
cells are not as clear, so it may be difficult to pick out
different cell types in a heterogeneous suspension on a scatter
plot. This isn't an issue for researchers like you that are
working with cell lines.
For a detailed copy of our protocol, please visit our website
(http://www.cellsignal.com), click on Support, then Research
Protocols, and finally on Flow. Please feel free to contact me
if you have any questions.
Best of luck,
--Randy Wetzel
CLB protocol for membrane and intracellular FACS staining : (By
Paul Baars, CLB-KVI) Reagents: PBS PBS 0.5% BSA PBS 0.1%
saponin 0.5% BSA Human Pooled Serum (HPS) 4% Paraformaldehyde
(PFA) in PBS Procedure (the whole procedure is performed on
ice)
1. Wash the cells in a 15 ml tube in PBS 0.5% BSA
2. Suspend the cells in PBS 0.5% BSA (4x106/ml) and add direct
conjugated Mab's for the membrane staining
3. Incubate for 30 min
4. Wash 1X with PBS 0.5% BSA
5. Wash 1X with PBS
6. Add 1.5 ml 4% PFA in PBS and incubate for 5 min (stopwatch!!)
7. Wash 1X with PBS
8. Wash 1X with PBS 0.1% saponin 0.5% BSA
9. Suspend the cells in PBS 0.1% saponin 0.5% BSA + 10% HPS
10. Incubate for 20 min
11. Wash 1X with PBS 0.1% saponin 0.5% BSA
12. (From now on this buffer is used until the end of the
procedure)
13. Suspend the cells in a x 50ml (a= number of different
intracellular staining) and pipette the cells in a 96-well dish
14. Add Mab's to the intracellular antigen and control Mab's
(diluted in saponin buffer)
15. Incubate for 30 min
16. Wash 3X 17. Measure the cells on the FACS We know that
other permeabilization procedures are also successful for
Granzyme staining e.g. the BFA fixation,
john voorn
For B27 as a PE or APC conjugate, we typically use 1 mcg/ml
final concentration. We titer every lot.
I don't know what the
stock concentration of your B27 mAb is, so if you want the exact
calculation you will have to do the calculation or send me the
concentration offline. A guess is that the stock concentration
is 200 mcg/ml (100 mcg in 500 uL) and you are using it at a
1:285 dilution, which gives a final concentration of 0.7 mcg/ml.
Looks pretty reasonable to me. A common mistake that is further
propagated by many of the Ab companies is to use antibodies by
mass as for example, requiring "0.5 mcg per test". The variable
that makes the difference is mAb concentration, not total
amount. We typically stain in 50 ul and thus "per test" only
requite 1/4 the total mass of mAb to maintain the same
concentration as you would when staining in 200 ul.
Calman Prussin Allergic Diseases Section NIAID/ NIH
Although I agree monensin is not the perfect blocker of
intracellular transport, I think your response is a bit
exaggerated. What toxicity data are you citing? The Jung paper
demonstrated monensin toxicity only after 16 hours. Both
monensin and BFA need only be in the culture for 2-4 hours for
maximal effect (my data and others). Also consider that the
strong stimuli (PMA/ionomycin) that are often used to effect
sufficient cytokine expression are also causing cell death. A
couple of hours of monensin may not be the largest perturbation
in the system. I have not compared a large number of cytokines,
but for huIL-2 and IFN I do not see a significant difference in
the mean fluorescence intensity or % positives between BFA and
monensin. The main reason for using monensin in the past was
that it was dramatically cheaper. Sigma now sells BFA at a
reasonable price. It is worth having a through discussion of
the relative merits of each, as I am eager to switch to a
better reagent. I look forward to hearing from you to
substantiate your statement. Intracellularly Backed Up in
Bethesda,
Calman Prussin Allergic Diseases Section NIAID/ NIH
The protocol that I find most useful for permeabilisation for
the detection of internal antigens is to use saponin. It is
relatively gentle (ie it doesn't put enormous holes in the
cytoplasm or the cell membrane) and can be used in conjunction
with surface staining or DNA staining. You may have to play
about with conditions but a good starting point is to treat
cells with 0.3% saponin for about 15mins at room temperature
before doing the antigen detection and then to use 0.1% saponin
in all subsequent steps. Be careful with the washing steps as
it is easy to lose all your cells! We have found that the
permeabilisation is best at room temperature and that the
saoponin needs to be present all the time as the
permeabilisation effect can be reversed. As with all
intracellular staining washing is important, but if you are
using directly conjugated antibody, this reduces the steps and
reduces cell loss. Hope that this is of some use.
Derek Davies Imperial Cancer Research Fund London
Hi Dr. Roy and fellow flowers: I do this all the time. I have
abandoned Western blot in favor of intracellular flow cytometry,
and I have compared indirect to direct staining, with similar
results. I use either a biotinylated first step or an unlabeled
first step. If I use an unlabeled, I use a ligand-affinitypurified polyclonal, and a labeled F(ab2') for the second step.
I block with Ig of the same species as the F(ab2'). I have
successfully seen p38, STAT3, JAK2, and a lot of cytokines. The
first step does not have to be one specifically for flow to
work--if it works in westerns and immunohistochemistry chances
are excellent that the Ab will work here too. A major pitfall is
not blocking sufficiently and not washing sufficiently. I block
with several mg/ml Ig for about 1 hr on ice after
permeabilization. Also, the first wash after adding Ig, Ab, or
second step must be with buffer added and left there for the
same length of time as the incubation step. For example, if you
left Ab on for 45 min, you must spin out the Ab and then leave
the wash buffer on for 45 min, then proceed with the subsequent
washes as usual. Another thing I found was that Caltag fix &
permeabilization reagents gave me superior signal to noise
ratios for phospho-Ab labeling, while Pharmingen or home-made
reagents gave me very good results for intracellular cytokines.
See these references for more details: Fleisher et al., Clin
Immunol 90:425-430, 1999 Barton & Murphy Cytokine 12:18-27, 2000
Feel free to contact me for more info. Best,
Beverly Barton
Assistant Professor Dept. of Surgery
polarisation
Maris Handley wrote- >Someone recently brought me an article
entitled: Fluorescence polarization >assay by flow >cytometry.
The authors are J.M. Rolland, K. Dimitropoulos, G.R. Hocking,
>and R.C. Naim. >The article is from 1985. My first question is
whether anyone is >routinely looking at >fluorescence
polarization in 2001? If not, is this because there is a >newer
method, >or better technology for looking at subtle cellular
changes? >I would be interested in talking to anyone who has
experience with these >kinds of >measurements. See pages 327-9
of the 3rd Edition of Practical Flow Cytometry for additional
background information. The fluorescence polarization assay in
question was based on observations made by Boris and Lea Cercek,
working in Manchester, England in the mid-1970's, that the
polarization of fluorescein fluorescence in lymphocytes
(produced by intracellular enzymatic hydrolysis of fluorescein
diacetate, also known as FDA) changed within a relatively short
time after mitogenic stimulation by phytohemagglutinin (PHA).
The measurements, described as showing changes in the
"structuredness of cytoplasmic matrix", or SCM, were originally
done on lymphocyte suspensions in a spectrofluorometer. The
Cerceks also reported that lymphocytes from patients with cancer
exhibited a diminished polarization response to PHA, but, unlike
patients without cancer, showed a polarization response to
proteins derived from cancer cells, and proposed this as a test
for cancer. While Rolland et al and others attempted to
implement the assay using flow cytometry, with varying degrees
of success, the most precise single cell fluorescence
polarization measurement system is the Cellscan, developed by
Prof. Mordechai (Motti) Deutsch and his colleagues at Bar Ilan
University in Israel. They have published numerous papers in
recent years (see bibliography below), and now believe that the
polarization changes reflect changes in the cytoskeleton and/or
intracellular hydration state. Motti Deutsch has criticized the
notion of measuring polarization in flow, because the constant
observation time means fewer photons are counted for cells with
weaker fluorescence, making measurements less precise.
Computation of the polarization value requires taking a ratio,
and the change in this ratio is relatively small, meaning that
any source of imprecision decreases the likelihood of obtaining
significant results. The Cellscan illuminates the cell with
very low intensity light (minimizing bleaching) for as long as
is necessary to count 10,000 photons in each of two
polarizations for each of two fluorescence wavelengths,
providing a very precise polarization measurement. I don't know
of recent data bearing on whether commercial flow cytometers are
up to the job. An overall problem with the polarization assay
is that the Cellscan apparatus, while precise for polarization
measurements, has not been equipped with sufficient
multiparameter measurement capability to allow determination of
other cellular characteristics, such as phenotype, activation
antigen expression, RNA or DNA content, cytoplasmic calcium
concentration, or membrane potential, which also reflect
lymphocyte activation. The differences in lymphocyte activation
patterns between patients with and without cancer that are
reported to be detected by the polarization assay should
presumably be detectable using more widely accepted indicators
of lymphocyte activation, but the necessary comparison
experiments remain to be done. I personally think this would be
worthwhile, because some of the results I have seen from both
the Cellscan and flow cytometric studies suggest that pursuing
this line of inquiry could yield some interesting new
information relevant to tumor immunology. Older references
cited in the book; the newer papers are: Deutsch M, Weinreb A:
An apparatus for high-precision repetitive sequential optical
measurement of living cells. Cytometry 1994; 16:214-26 Ron IG,
Deutsch M, Tirosh R, Weinreb A, Eisenthal A, Chaitchik S:
Fluorescence polarisation changes in lymphocyte cytoplasm as a
diagnostic test for breast carcinoma. Eur J Cancer 1995;
31A:917-20 Eisenthal A, Marder O, Dotan D, Baron S, LifschitzMercer B, Chaitchik S, Tirosh R, Weinreb A, Deutsch M Decrease
of intracellular fluorescein fluorescence polarization (IFFP) in
human peripheral blood lymphocytes undergoing stimulation with
phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed
mitogen (PWM) and anti-CD3 antibody. Biol Cell 1996; 86:145-50
Marder O, Shoval S, Eisenthal A, Fireman E, Skornick Y,
Lifschitz-Mercer B, Tirosh R, Weinreb A, Deutsch M: Effect of
interleukin-1 alpha, interleukin-1 beta and tumor necrosis
factor-alpha on the intracellular fluorescein fluorescence
polarization of human lung fibroblasts. Pathobiology 1996;
64:123-30 Deutsch M, Ron I, Weinreb A, Tirosh R, Chaitchik S:
Lymphocyte fluorescence polarization measurements with the
cellscan system: application to the SCM cancer test. Cytometry
1996; 23:159-65 Eisenthal A, Marder O, Lifschitz-Mercer B,
Skornick Y, Tirosh R, Weinreb A, Deutsch M Inhibition of
mitogen-induced changes in intracellular fluorescein
fluorescence polarization of human peripheral blood lymphocytes
by colchicine, vinblastine and cytochalasin B. Cell Struct Funct
1996; 21:159-66 Zurgil N, Deutsch M, Tirosh R, Brodie C:
Indication that intracellular fluorescence polarization of T
lymphocytes is cell cycle dependent. Cell Struct Funct 1996;
21:271-6 Rahmani H, Deutsch M, Ron I, Gerbat S, Tirosh R,
Weinreb A, Chaitchik S, Lalchuk S: Adaptation of the cellscan
technique for the SCM test in breast cancer. Eur J Cancer
1996;32A:1758-65; Comment in: Eur J Cancer. 1997; 33:1333-5
Merimsky O, Kaplan B, Deutsch M, Tirosh R, Weinreb A, Chaitchik
S: Detection of melanoma by monitoring the intracellular
fluorescein fluorescence polarization changes in lymphocytes.
Cancer Detect Prev 1997; 21:167-77 Eisenthal A, Marder O,
Lifschitz-Mercer B, Skornick Y, Fixler D, Avtalyon R, Tirosh R,
Deutsch M: Influenza A virus affects the response of human
peripheral blood mononuclear cells to phytohaemagglutinin A by
altering the cytoskeleton. Pathobiology 1997;65:69-74 Sunray M,
Deutsch M, Kaufman M, Tirosh R, Weinreb A, Rachmani H: Cell
activation influences cell staining kinetics Spectrochim Acta A
Mol Biomol Spectrosc 1997;53A:1645-53 Cohen-Kashi M, Deutsch M,
Tirosh R, Rachmani H, Weinreb A: Carboxyfluorescein as a
fluorescent probe for cytoplasmic effects of lymphocyte
stimulation.Spectrochim Acta A Mol Biomol Spectrosc 1997;
53A:1655-61 Eisenthal A, Marder O, Lifschitz-Mercer B, Skornick
Y, Tirosh R, Irlin Y, Avtalion R, Deutsch M: Infection of K562
cells with influenza A virus increases their susceptibility to
natural killer lysis. Pathobiology 1997; 65:331-40 Fixler D,
Tirosh R, Eisenthal A, Marder O, Irlin Y, Lalchuk S, Deutsch M:
Monitoring of effector and target cell stimulation during
conjugation by fluorescence polarization. Biol Cell 1997;
89:443-52 Gelman-Zhornitsky E, Deutsch M, Tirosh R, Yishay Y,
Weinreb A, Shapiro HM: 2, 7'- bis-(carboxyethyl)-5-(6)carboxyfluorescein (BCECF) as a probe for intracellular
fluorescence polarization measurements. J Biomed Optics. 1997;
2:186-194 Avtalion N, Avtalion R, Tirosh R, Sheinberg A,
Weinreb A, Avinoach I, Deutsch M: Preparation of a diagnostic
antigen of human melanoma based on lymphocyte activation as
measured by intracellular fluorescein fluorescence polarization.
Cancer Detect Prev 1999; 23:64-71 Sunray M, Kaufman M, Zurgil
N, Deutsch M: The trace and subgrouping of lymphocyte activation
by dynamic fluorescence intensity and polarization measurements.
Biochem Biophys Res Commun 1999; 261:712-9 Zurgil N, Levy Y,
Deutsch M, Gilburd B, George J, Harats D, Kaufman M, Shoenfeld
Y: Reactivity of peripheral blood lymphocytes to oxidized lowdensity lipoprotein: a novel system to estimate atherosclerosis
employing the Cellscan. Clin Cardiol 1999; 22:526-32 Zurgil N,
Kaufman M, Solodiev I, Deutsch M: Determination of cellular
thiol levels in individual viable lymphocytes by means of
fluorescence intensity and polarization. J Immunol Methods
1999; 229:23-34 Zurgil N, Schiffer Z, Shafran Y, Kaufman M,
Deutsch M: Fluorescein fluorescence hyperpolarization as an
early kinetic measure of the apoptotic process. Biochem Biophys
Res Commun 2000; 268:155-63 Deutsch M, Zurgil N, Kaufman M,
Berke G: Fluorescence polarization as an early measure of Tlymphocyte stimulation.Methods Mol Biol 2000; 134:221-42
eutsch M, Kaufman M, Shapiro H, Zurgil N: Analysis of enzyme
kinetics in individual living cells utilizing fluorescence
intensity and polarization measurements. Cytometry 2000;39:36-44
-Howard
software crashes
Things that will make your Calibur Mac Crash:
1. Directly saving data to a peripheral (zip, memory stick,
external hard drive). Save it to the HD and transfer after
acquisition is complete. Remove peripheral memory device while
acquiring. That USB connection isn't as fast as the computer is
and so things lock up.
2. Connecting to network during acquisition. Turn off file
sharing in control panels or disconnect the mac from the network
completely. Try setting tcp/ip setting in control panels to
"only when needed."
3. Computer is set to sleep. Change sleep setting to "never" in
energy saver control panel.
4. A mouse with a damaged cord. Replace the mouse.
5. If you use FileGuard, making new folder from inside CQ
(folder button in Parameter Description window). Make new
folder in desired location before opening CQ, then navigate to &
choose your folder in CQ.
6. Multi-tasking while acquiring. Specifically, avoid RAM
intensive applications.
Things to prevent a Calibur Mac Crash:
1. Rebuild the HardDrive. Hold down "Option" and "Apple" keys
while restarting computer.
2. De-frag the HardDrive. I use Norton Disk Dr at least twice
a month and run the diagnostic and de-fragment the HD.
3. Don't use PC formatted zip (problematic for those users from
PC based labs).
4. Don't analyze data on acquisition machines. 5. Keep your HD
less than 50% full. 6. Stay away from USB hubs if possible. 7.
Zap the P-RAM. This will re-set your preferences. Hold down
"Option," "Apple," "P," and "R" keys on restart.
The appearance of the sub-G0/G1 is highly variable between cell
types, and even the same cell type at differing levels of
activation. Cycling cells in particular (with less compacted
chromatin) are particularly likely to give confusing results,
especially with the accumulation of subcellular objects
(cell fragments, fragmented chromatin, other trash) at the
low threshold of instrument sensitivity. The contribution
of S + G2/M cell apoptosis to the "pot" further complicates the
issue of
data interpretation. And if we can't clearly separate intact
apoptotic cells from apoptosis-associated debris, we are not
really measuring
cell-by-cell apoptosis anymore.
We strongly encourage our investigators to use sub-G0/G1 ONLY as
a
preliminary, qualitative indicator of cell death, not as a
quantitative assay - unless the apoptotic peak is really clear,
which does not
happen most of the time. It should always be backed up with
additional
assays, especially biochemical ones like caspase activation. And
most
journal reviewers, grant review panels, etc. won't be impressed
anymore if DNA loss is the only criterion for measuring cell
death - there are
lots of other nice flow apoptosis assays available now, some of
them fairly
economical.
Enjoy,
Bill Telford
I have not had any problems (background staining) with mouse
MEFs so far. For routine Fc blocking when using mouse tissue
samples, I incubate the cells with 2.4G2 (anti-Fc) for 20-30
minutes on ice. Usually, I do not even wash the cells after
blocking (a quick spin for removing 2.4G2 supernatant). It
works really well for me. Ocassionally, in my liver MNC
preparations, I do get background B cell staining inspite of
2.4G2 block.
I have not got around this problem yet (other
than using a negative B220 gate).
I have not used the Fc block after the first blocking step. I
block the cells first with the anti-Fc antibody, surface stain
and directly fix and permeabilize my cells for intracellular
staining. Hope this helps.
Sriram
Venkataraman Sriram, PhD
Postdoctoral Fellow
The Walther Cancer Institute
Indiana University School of Medicine
Indianapolis, IN 46202
viability
Thanks. You are right ! I was using different definition of
viability - > the pragmatic rather than the logical one. I
defined cell viability in > terms of survival of tumor cells
treated with antitumor drugs - are they > later able to
proliferate/ form colonies ?. Since I am working on > antitumor
drugs/strategies, I must developed such "tunel vision" in >
interpreting live vs dead cells (identifying reproductive cell
death with > cell death) > Regards > Zbigniew
Robert, Mitotracker Red CMXRos remains in mitochondria after
cell fixation/permeabilization. We do not have experience with
Mitotracker Green but Haugland makes note in his Handbook that
it does not remain in the cell after fixation/permeabilization.
Using non-fixed, live, cells we were able to measure Mitotracker
Red, as well as tetramethylrhodamine methyl ester (TMRM, Sigma)
fluorescence, each in combination with FLICA (Fig 3 in our
recent Cytometry paper), using just 488 nm excitation, in
FacsScan. Vermuellen et al., (Exp. Hematol 30, 1107-1114, 2002)
also had nice results with TMRM e.g in combination with Annexin
V using FaacsScan. We did not check whether TMRM can be fixed.
Best wishes and regards Zbigniew
Dear All, Neither MTT nor PI is a reliable assay of cell
viability. The PI assay is based on the detection of the loss of
plasma membrane capability to exclude this dye. Early apoptotic
cells exclude PI. Only necrotic and late apoptotic cells stain
with PI. Thus, early apoptotic cells, although for all practical
reasons they are dead (certainly reproductively dead), are
recognized as live cells by this assay. On the other hand MTT
assay measures the "cell redox activity", that is to a large
extent mitochondrial but may also be non-mitochondrial (e.g. see
Bernas and Dobrucki, Cytometry, 2002;47:236-242). The agents
that arrest the cell cycle progression (e.g activating cell
cycle checkpoints, inhibitors of DNA polymerase etc) induce
unbalanced cell growth. In the absence of DNA replication the
cells grow in size, including the increase in mitochondrial mass
and activity. Such cells are are moribound - at certain degree
of growth unbalance they are irreversibly commited to die. By
the MTT assay, however, not only such cells are detectable as
live, but with time one sees their increased capacity to reduce
MTT. It may appear, therefore that cells in cultures proliferate
(this can be seen for up to three days), whereas in fact they
are reproductively dead. One has to note that some vendors
advertise the MTT kits as "Cell Proliferation Assays", which is
quite misleading. The gold standard in viability assays is
clonogenicity test. Unfortunately it is cumbersome and time
consuming. With each new drug/cell system measured by MTT or
other rapid/automatic assays, it is advised to run at least once
the clonogenicity test, for comparison.
Zbigniew Darzynkiewicz, M.D., Ph.D.
This is a fabulous way to do viability testing! Once you do
this method, you will never do a trypan blue (yech) again. I
learned to do this in the Herzenberg laboratory at Stanford,
brought it to the VRC--and we've now incorporated it in our
clinical trials. Every time we thaw PBMC for doing immune
function assays, we assess the viability by fluorescence first
(and, in fact, if viability is below a treshhold, I think 60%,
we discard the sample). We've even developed an SOP for it. We
use a combination of acridine orange and ethidium bromide (not
PI)--under a fluorescence scope, "green" is live and "red" is
dead--no ifs, ands, or buts--and easily scored by even the most
green students with risking a red face. In any case, our
procedure is to prepare 3 mg/ml ethidium bromide in absolute
ethanol and 5 mg/ml acridine orange in ethanol. Store this
stock in a dark vial, refrigerated. To make a working solution,
take 1 microliter of each added to 1 milliliter of PBS. This we
store at room temp by the fluorescence microscope, and make
fresh every few weeks. Please note that AO and EB are
considered highly carcinogenic: use gloves and a face mask when
preparing the concentrated stock solution, and use gloves when
handling the working solution. Dilute cells with an equal
volume of the working solution and immediately look on the
fluorescence microscope (you can also dilute 1:10 if the cell
count is too high). Remember to take this dilution into account
when you calculate original numbers.
EB, you could
mr
(PS, if you don't have
I hesitate to disagree with Maryalice, especially given her
powerful admonition that whoever says otherwise knows absolutely
nothing about PI exclusion... but... "he who hesitates is
last". Actually, we found that we can stain with PI, then fix
with 0.5% paraformaldehyde, and have the ability to discriminate
live/dead for about 2 hours afterward (perhaps as long as 4-6
hours). Waiting overnight, however, is right out--the PI leaks
out of dead cells (and, if present in the medium, leaks into
live cells). As Mark points out, you should add the PI before
the PF, and if you need to wait more than several hours, use EMA
(which is considerably less practical for various reasons). We
tested this extensively, because of the importance of doing
live/dead discrimination, as well as the practicality of fixing
cells (for example, from infectious samples). Note that we did
not test higher concentrations of paraformaldehyde or other
fixatives. mr (PS, with regard to removal of adherent
endothelial or tumor cells becoming PI+ : note that a variety of
protocols, as asserted already on this list, can transiently
permeabilize cells. I would try removing the cells, washing
them well in regular medium, waiting 30 minutes, and then adding
PI).