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. mr (PS, if you don't have
EB, you could
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).