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 five- fold 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