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A new assay for cell death
Journal of Immunological Methods, 134 (1990) 201-205 Elsevier
201
JIM05749
A n e w assay for cell death Igor P. Beletsky a n d Samuil R. U m a n s k y Institute of Biological Physics, U.S.S.R. Academy of Sciences, Pushchino, Moscow Region, 142292, U.S.S.R. (Received 9 January 1990, revised received 30 April 1990, accepted 9 August 1990)
A new assay for the evaluation of cell viability is described. It is based on the staining of dead cells and subsequently the whole cell population with ethidium bromide (EtBr). The method makes it possible to measure cytotoxic and cytostatic effects simultaneously and cell cultures grown both in suspension as well as by adherence may be assessed. The advantages and disadvantages of this assay are discussed. Key words: Cell death; Cytotoxic and cytostatic effects; TNF; Ethidium bromide fluorescence
Introduction
The determination of cytolytic and cytostatic effects of physical, chemical or biological agents is an important investigation in fields such as experimental immunology, pharmacology and oncology. To analyse cell viability, methods based on changes in external membrane permeability, registered by supravital dyes or 51Cr release, and on the conversion of vital dyes into a detectable compound in the living cell, are most widely employed (Duke et al., 1983; Nissen-Meyer et al., 1987; Meager et al., 1989). The cytostatic effect of various agents is usually estimated from the incorporation of labelled precursors into DNA, the change in the cell biomass or by flow cytometry (Johnson et al., 1979; Hacket et al., 1988) and the use of these techniques continues because of their simplicity and their satis-
Correspondence to: I. Beletsky, Institute of Biological Physics, U.S.S.R. Academy of Sciences, Pushchino, Moscow Region, 142292, U.S.S.R. Abbreviations: EtBr, ethidium bromide; rhTNF, recombinant human tumor necrosis factor; AcD, actinomycin D.
factory reproducibility. However, there are a number of limitations to such assays. For example, similar numbers of living cells, as determined by staining with vital dyes in both control and test samples, are usually interpreted as the lack of an effect, but the same results can be obtained if the incubation time is long enough and the rate of proliferation significantly exceeds that of cell death (Lynch et al., 1986). A decrease in the number of cells stained by crystal violet is generally taken to be the result of a cytotoxic effect. However, one cannot exclude the possibility that such a result may be the consequence of a cytostatic effect on dividing cells in culture. Thus, evaluation by the registration of living or dead cells alone is only correct with respect to resting cells. Furthermore the quantitation of a cytotoxic effect determined by MTT or crystal violet staining is generally overestimated because dead cells, usually present in the control, are not taken into account. Finally for the employment of some dyes, for instance crystal violet, it is necessary to remove dead cells, which makes it impossible to analyse suspension and semisuspension cell cultures. Here we describe a simple method for the evaluation of cell viability which is intended for the automatic scanning of microwell plates and is
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
202
free of the above-mentioned limitations. First, EtBr is added to the cells and dead cell fluorescence measured. Then living cells are permeabilised with digitonin and the fluorescence measurement is repeated. The ratio of the first and second values obtained is proportional to the number of dead cells. A similar approach has been used for the evaluation of permeable cells by flow cytometry (Afanasiev et al., 1988).
A ssay Cytotoxicity was assayed as follows: 10/~1 of 20 /~g/ml EtBr were added to each well with control or treated cells and after incubation for one hour the fluorescence of dead cells was measured at 610 nm. Then digitonin was added to the medium (final concentration of 50/~g/ml) and I h later the measurement was repeated. The percentage cyto-
A
Materials and m e t h o d s 1000
Cell cultures The L 929, murine fibrosarcoma cell line was kindly provided by Dr. Zemskova, IBPhM, U.S.S.R. Academy of Sciences, Pushchino. It was maintained in a monolayer culture in Eagle's medium containing 10 % fetal calf serum, 2 mM L-glutamine, 100 U / m l penicillin and 100 /xg/ml streptomycin. Thyrnocytes from male Wistar rats of 150-160 g were prepared as described (Beletsky et al., 1989) and were maintained in RPMI 1640 medium supplemented with penicillin, streptomycin, g-glutamine and 25 mM Hepes. Cell viability determinations The activity of r h T N F (kindly provided by Dr. S. Nedospasov, IMB, U.S.S.R. Academy of Sciences, Moscow) was measured on actinomycin-Dtreated murine L 929 cells as described (Kronke et al., 1988). Briefly, a cytotoxic assay measured at 18 h was performed by seeding 96 well plates with 20 x 103 cells/well in 0.1 ml and allowing them to adhere overnight at 37°C. Serial dilutions of r h T N F in Eagle's medium with AcD (0.5 /~g/ml) were added in 0.1 ml aliquots and the plates incubated for 18 h at 37°C. Then medium was removed and cell lysis was determined by staining the cells with crystal violet (0.5%) in m e t h a n o l / water (1:4, v/v), followed by automatic plate reading at 540 nm. Thymocytes (107 cell/ml) were irradiated with 6°Co at a dose of 10 Gy (dose rate 2.72 G y / m i n ) . A cell viability assay was performed by seeding 96 well plates with 30 × 103 cells/well. After 7 h of incubation at 37 ° C, trypan blue was added and the stained cells were counted by microscopic observation (Kawakami et al., 1989).
500
0i ! B
s
i 1000 t
500
0
3
400
, 50O A Into)
. 6OO
Fig. 1. Fluorescence spectra of microwell plates 'Nunc' (1), 'Novogene' (2), 'Titertek' (3,4) at exciting wave-lengths of 365 nm (A) and 300 n m (B); 4: EtBr was added to the incubation medium. Abscissa: wavelength (nm); ordinate: fluorescence in relative units. Fluorescence in the experiments presented in Fig. 1A and Fig. 1B was registered in different regimes of the photomultiplier.
203
toxicity was determined according to the following equation: % cytotoxicity
F 1 - Fo F2 _ Fo
F I ' - Fo ~
x
100%
(1)
where F 1, F ( represent the fluorescence of dead cells in experimental and control wells, respectively; F2, F 2' represent the fluorescence of the whole cell population in experimental and control wells, respectively and F 0 is the EtBr fluorescence in the absence of cells. The cytostatic effect was evaluated by comparison of F 2 and F2'. Measurements were carried out with a spectrophotometer-densitometer SD-1 (Jiromsky et al., 1978) employing transmitted light. A beam diameter equal to that of the well (6 mm) was generally used to analyse the cells.
Results
Spectral properties of plates Since the method was based on the measurement of fluorescence by cells stained in plates with EtBr, it was necessary to study the fluorescent properties of the plates. Initially it was found that the plates had intrinsic fluorescence at the exciting wave-length 2~0 = 365 nm, the fluorescence spectrum of EtBr being significantly overlapped by that of the plates (Fig. 1A). We attempted to
70C
500 L
o _u
6(3
300
LL
lOO
4C
°
20
0,,,, ll2
~
- 11
i
- 10
,
- 9
J ,
- 8
Log [TNF] M Fig. 3. Dependence of cytotoxicity on T N F concentration. Cytotoxicity was measured by crystal violet (©) and by EtBr (o) (see Materials and Methods section).
reduce the emission of plates at 610 nm by changing the exciting wave-length )'0 and it was found that a shift towards the short-wave region resulted in fluorescence lowering, and at 2~0 = 300 nm the plate fluorescence was negligible in the region of EtBr fluorescence (Fig. 1B). From a comparison of the spectral properties of plates produced by 3 different manufacturers Nunc (Denmark), Novogene (C.S.S.R.) and Flow (U.K.) - it is clear (Fig. 1) that they all contain fluorophor(s) with a fluorescence maximum )kma x = 410 nm. The apparent shift of X,,ax accompanying the change of the exciting wave length is accounted for by the presence of cut-off filters in the registering channel of the fluorometer. 'Nunc' plates showed the least emission and 'Flow' plates gave the largest. In preliminary experiments it was shown that EtBr emission was directly proportional to the number of added cells (Fig. 2), establishing that it is possible to use this assay for the quantitative measurement of cytotoxicity. It was noted that bubbles on the medium surface resulted in a disturbance of the fluorescence measurements and it was necessary therefore that these be previously removed.
, 0
10
30
50
70
90
× 103
Cell n u m b e r
Fig. 2. Relationship between cell number and fluorescence intensity. Different numbers of L 929 cells were seeded in the wells of a 96-well plate, digitonin and EtBr were added, and fluorescence was measured 1 h later.
Analysis of TNF and radiation cytotoxicity Two model systems, TNF-dependent lysis of L 929 cells and death of irradiated thymocytes, were used. L 929 cells were plated overnight at 2 x 10 4 cells/well in 100 /~1 of medium containing 10%
204 40
3O u
o
20
10
Fig. 4. The effect of 3,-irradiation (10 Gy) on rat thymocyte viability. Cytotoxicity was measured by EtBr (A) and trypan blue (B) staining. White bars = control; black bars = irradiated thymocytes.The data represent the mean of 3 assays. fetal calf serum. Then the medium was replaced with one containing AcD and T N F without serum. After 18 h of incubation, cell lysis was determined by crystal violet staining (see Materials and Methods section) and by the proposed method. One can see from Fig. 3 that the results obtained by both methods were quite similar and in accordance with the literature (Coffman et al., 1988; Mace et al., 1988). We further verified the applicability of the method for the evaluation of cell death in suspension. Isolated rat thymocytes were irradiated and after 7 h of incubation the percentage of dead cells was determined. For comparison cells stained with trypan blue were counted by microscopy. Figure 4 shows that the results were essentially the same and analogous to the radiobiological data (Afanasiev et al., 1988).
specific fluorescent dyes, such as Hoechst 33258, DAPI and EtBr, only EtBr is satisfactory for the automatic scanning of plates. Not only do Hoechst 33258 and D A P I penetrate living cells slowly, but their fluorescence spectra are significantly overlapped by that of the plates (~ .... = 505 and 465 nm, respectively). In this study the exciting wavelength of 300 nm was chosen for the following reasons. First, it is one of the optimum exciting wavelengths for EtBr (Osterman, 1984); second, at this 2% value the emission of plates at 610 nm is negligible (Fig. 1) and, finally, such a wavelength is provided in some automatic readers (for example, Fluoroscan II, Flow). The proposed method has some further advantages over other techniques. First, the preliminary separation of dead and living cells is not obligatory. As stated above this simplifies the procedure and also makes it possible to evaluate both the cytotoxic and cytostatic effects simultaneously. Second, cell debris, formed during long incubations and undetectable by method other than flow cytometry (Deitch, 1987), can be taken into account in this system. Third, the consecutive staining of dead cells and subsequently the whole population permits one to determine the proportion of dead cells in the control samples. Thus an evaluation of absolute cytolysis values becomes possible. Usually such values (the actual value of the test sample minus that of the control sample) are lower than those obtained, for example, by crystal violet staining (Fig. 3). In addition it should be noted that the proposed method is applicable to the analysis of both dividing and quiescent cells as well as plated and suspension cell cultures.
Discussion In some circumstances cytotoxicity cannot be correctly determined by the automatic scanning of plates with cells stained by traditional methods, since vital and supravital dyes differ in their properties. It would be advantageous to use a compound possessing properties of both vital and supravital dyes and some fluorescent dyes exhibit such properties. They are able, on one hand, to enter dead cells and, on the other hand, their emission increases markedly on binding to nucleic acids. However, among widely used nucleic acid-
Acknowledgement We would like to thank V. Shlektarev for technical assistance.
References Afanasiev, V.N., Korol, B.A., Matylevich, N.P., Pechatnikov, V.A., Sorokina, N.I., Umansky, S.R. and Filippovich, l.V. (1988) General regularities and variations in the postirradi-
205 ation death rate of lymphocytes of various animal species and man. Radiobiologiya (U.S.S.R.) 28, 731. Beletsky, I.P., Matyasova, J., Nikonova, L.V., Scalka, M. and Umansky, S.R. (1989) On the role of Ca,Mg-dependent nuclease in the postirradiation degradation of chromatin in lymphoid tissues. Gen. Physiol. Biophys. 8, 381. Coffman, F.D., Green, L.M. and Ware, C.F. (1988) The relationship of receptor occupancy to the kinetics of cell death mediated by tumour necrosis factor. Lymphokine Res. 7, 371. Deitch, A.D. (1987) The effect of cytotoxicity on DNA histograms. Cytometry 8, 339. Duke, R.C., Chervenak, R. and Cohen, J.J. (1983) Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. J. Immunol. 80, 6361. Hacket, R.J., Davis, L.S. and Lipsky, P.P. (1988) Comparative effects of tumour necrosis factor-a and IL-lfl on mitogeninduced T cell activation. J. Immunol. 140, 2639. Jiromsky, V.V., Medvedev, A.I., Schichkov, V.V., Medvedev, B.I., Ivanov, E.G. and Schirogorov, A.A. (1978) Spectrofotometer-densitometer. Pribori Tekhn. Eksp. (U.S.S.R.) 6, 204. Johnson, L.K., Lan, N.C. and Baxter, J.D. (1979) Stimulation and inhibition of cellular function by glucocorticoids, J. Biol. Chem. 254, 7785. Kawakami, M., Watanabe, N., Ogawa, H., Kato, A., Sando,
H., Yamada, N., Murase, T., Takaku, F., Shibata, S. and Oda, T. (1989) Cachectin/TNF kills or inhibits the differentiation of 3T3-L1 cells according to developmental stage. J. Cell. Physiol. 138, 1. Kronke, M., Hensel, G., Schluter, C., Scheurich, P., Schutze, S. and Pfizenmaier, K. (1988) Tumour necrosis factor and lymphotoxin gene expression in human tumour cell lines. Cancer Res. 48, 5417. Lynch, M.P., Nawaz, S. and Gerschenson, L.E. (1986) Evidence for soluble factors regulating cell death and cell proliferation in primary cultures of rabbit endometrial cells grown on collagen. Proc. Natl. Acad. Sci. U.S.A. 83, 4784. Mace, K.F., Ehrke, M.J., Hod, K., Maccubbin, D.L. and Mihich, E. (1988) Role of tumour necrosis factor in macrophage activation and tumoricidal activity. Cancer Res. 48, 5427. Meager, A., Leung, H. and Woolley, J. (1989) Assays for tumour necrosis factor and related cytokines. J. Immunol. Methods 116, 1. Nissen-Meyer, J., Austgulen, R. and Espevik, T. (1987) Comparison of recombinant tumour necrosis factor and the monocyte derived cytotoxic factor involved in monocytemediated cytotoxicity. Cancer Res. 47, 2251. Osterman, L.A. (1984) In: Methods of Protein and Nucleic Acids Research. Springer Verlag, Berlin, p. 150.
201
JIM05749
A n e w assay for cell death Igor P. Beletsky a n d Samuil R. U m a n s k y Institute of Biological Physics, U.S.S.R. Academy of Sciences, Pushchino, Moscow Region, 142292, U.S.S.R. (Received 9 January 1990, revised received 30 April 1990, accepted 9 August 1990)
A new assay for the evaluation of cell viability is described. It is based on the staining of dead cells and subsequently the whole cell population with ethidium bromide (EtBr). The method makes it possible to measure cytotoxic and cytostatic effects simultaneously and cell cultures grown both in suspension as well as by adherence may be assessed. The advantages and disadvantages of this assay are discussed. Key words: Cell death; Cytotoxic and cytostatic effects; TNF; Ethidium bromide fluorescence
Introduction
The determination of cytolytic and cytostatic effects of physical, chemical or biological agents is an important investigation in fields such as experimental immunology, pharmacology and oncology. To analyse cell viability, methods based on changes in external membrane permeability, registered by supravital dyes or 51Cr release, and on the conversion of vital dyes into a detectable compound in the living cell, are most widely employed (Duke et al., 1983; Nissen-Meyer et al., 1987; Meager et al., 1989). The cytostatic effect of various agents is usually estimated from the incorporation of labelled precursors into DNA, the change in the cell biomass or by flow cytometry (Johnson et al., 1979; Hacket et al., 1988) and the use of these techniques continues because of their simplicity and their satis-
Correspondence to: I. Beletsky, Institute of Biological Physics, U.S.S.R. Academy of Sciences, Pushchino, Moscow Region, 142292, U.S.S.R. Abbreviations: EtBr, ethidium bromide; rhTNF, recombinant human tumor necrosis factor; AcD, actinomycin D.
factory reproducibility. However, there are a number of limitations to such assays. For example, similar numbers of living cells, as determined by staining with vital dyes in both control and test samples, are usually interpreted as the lack of an effect, but the same results can be obtained if the incubation time is long enough and the rate of proliferation significantly exceeds that of cell death (Lynch et al., 1986). A decrease in the number of cells stained by crystal violet is generally taken to be the result of a cytotoxic effect. However, one cannot exclude the possibility that such a result may be the consequence of a cytostatic effect on dividing cells in culture. Thus, evaluation by the registration of living or dead cells alone is only correct with respect to resting cells. Furthermore the quantitation of a cytotoxic effect determined by MTT or crystal violet staining is generally overestimated because dead cells, usually present in the control, are not taken into account. Finally for the employment of some dyes, for instance crystal violet, it is necessary to remove dead cells, which makes it impossible to analyse suspension and semisuspension cell cultures. Here we describe a simple method for the evaluation of cell viability which is intended for the automatic scanning of microwell plates and is
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
202
free of the above-mentioned limitations. First, EtBr is added to the cells and dead cell fluorescence measured. Then living cells are permeabilised with digitonin and the fluorescence measurement is repeated. The ratio of the first and second values obtained is proportional to the number of dead cells. A similar approach has been used for the evaluation of permeable cells by flow cytometry (Afanasiev et al., 1988).
A ssay Cytotoxicity was assayed as follows: 10/~1 of 20 /~g/ml EtBr were added to each well with control or treated cells and after incubation for one hour the fluorescence of dead cells was measured at 610 nm. Then digitonin was added to the medium (final concentration of 50/~g/ml) and I h later the measurement was repeated. The percentage cyto-
A
Materials and m e t h o d s 1000
Cell cultures The L 929, murine fibrosarcoma cell line was kindly provided by Dr. Zemskova, IBPhM, U.S.S.R. Academy of Sciences, Pushchino. It was maintained in a monolayer culture in Eagle's medium containing 10 % fetal calf serum, 2 mM L-glutamine, 100 U / m l penicillin and 100 /xg/ml streptomycin. Thyrnocytes from male Wistar rats of 150-160 g were prepared as described (Beletsky et al., 1989) and were maintained in RPMI 1640 medium supplemented with penicillin, streptomycin, g-glutamine and 25 mM Hepes. Cell viability determinations The activity of r h T N F (kindly provided by Dr. S. Nedospasov, IMB, U.S.S.R. Academy of Sciences, Moscow) was measured on actinomycin-Dtreated murine L 929 cells as described (Kronke et al., 1988). Briefly, a cytotoxic assay measured at 18 h was performed by seeding 96 well plates with 20 x 103 cells/well in 0.1 ml and allowing them to adhere overnight at 37°C. Serial dilutions of r h T N F in Eagle's medium with AcD (0.5 /~g/ml) were added in 0.1 ml aliquots and the plates incubated for 18 h at 37°C. Then medium was removed and cell lysis was determined by staining the cells with crystal violet (0.5%) in m e t h a n o l / water (1:4, v/v), followed by automatic plate reading at 540 nm. Thymocytes (107 cell/ml) were irradiated with 6°Co at a dose of 10 Gy (dose rate 2.72 G y / m i n ) . A cell viability assay was performed by seeding 96 well plates with 30 × 103 cells/well. After 7 h of incubation at 37 ° C, trypan blue was added and the stained cells were counted by microscopic observation (Kawakami et al., 1989).
500
0i ! B
s
i 1000 t
500
0
3
400
, 50O A Into)
. 6OO
Fig. 1. Fluorescence spectra of microwell plates 'Nunc' (1), 'Novogene' (2), 'Titertek' (3,4) at exciting wave-lengths of 365 nm (A) and 300 n m (B); 4: EtBr was added to the incubation medium. Abscissa: wavelength (nm); ordinate: fluorescence in relative units. Fluorescence in the experiments presented in Fig. 1A and Fig. 1B was registered in different regimes of the photomultiplier.
203
toxicity was determined according to the following equation: % cytotoxicity
F 1 - Fo F2 _ Fo
F I ' - Fo ~
x
100%
(1)
where F 1, F ( represent the fluorescence of dead cells in experimental and control wells, respectively; F2, F 2' represent the fluorescence of the whole cell population in experimental and control wells, respectively and F 0 is the EtBr fluorescence in the absence of cells. The cytostatic effect was evaluated by comparison of F 2 and F2'. Measurements were carried out with a spectrophotometer-densitometer SD-1 (Jiromsky et al., 1978) employing transmitted light. A beam diameter equal to that of the well (6 mm) was generally used to analyse the cells.
Results
Spectral properties of plates Since the method was based on the measurement of fluorescence by cells stained in plates with EtBr, it was necessary to study the fluorescent properties of the plates. Initially it was found that the plates had intrinsic fluorescence at the exciting wave-length 2~0 = 365 nm, the fluorescence spectrum of EtBr being significantly overlapped by that of the plates (Fig. 1A). We attempted to
70C
500 L
o _u
6(3
300
LL
lOO
4C
°
20
0,,,, ll2
~
- 11
i
- 10
,
- 9
J ,
- 8
Log [TNF] M Fig. 3. Dependence of cytotoxicity on T N F concentration. Cytotoxicity was measured by crystal violet (©) and by EtBr (o) (see Materials and Methods section).
reduce the emission of plates at 610 nm by changing the exciting wave-length )'0 and it was found that a shift towards the short-wave region resulted in fluorescence lowering, and at 2~0 = 300 nm the plate fluorescence was negligible in the region of EtBr fluorescence (Fig. 1B). From a comparison of the spectral properties of plates produced by 3 different manufacturers Nunc (Denmark), Novogene (C.S.S.R.) and Flow (U.K.) - it is clear (Fig. 1) that they all contain fluorophor(s) with a fluorescence maximum )kma x = 410 nm. The apparent shift of X,,ax accompanying the change of the exciting wave length is accounted for by the presence of cut-off filters in the registering channel of the fluorometer. 'Nunc' plates showed the least emission and 'Flow' plates gave the largest. In preliminary experiments it was shown that EtBr emission was directly proportional to the number of added cells (Fig. 2), establishing that it is possible to use this assay for the quantitative measurement of cytotoxicity. It was noted that bubbles on the medium surface resulted in a disturbance of the fluorescence measurements and it was necessary therefore that these be previously removed.
, 0
10
30
50
70
90
× 103
Cell n u m b e r
Fig. 2. Relationship between cell number and fluorescence intensity. Different numbers of L 929 cells were seeded in the wells of a 96-well plate, digitonin and EtBr were added, and fluorescence was measured 1 h later.
Analysis of TNF and radiation cytotoxicity Two model systems, TNF-dependent lysis of L 929 cells and death of irradiated thymocytes, were used. L 929 cells were plated overnight at 2 x 10 4 cells/well in 100 /~1 of medium containing 10%
204 40
3O u
o
20
10
Fig. 4. The effect of 3,-irradiation (10 Gy) on rat thymocyte viability. Cytotoxicity was measured by EtBr (A) and trypan blue (B) staining. White bars = control; black bars = irradiated thymocytes.The data represent the mean of 3 assays. fetal calf serum. Then the medium was replaced with one containing AcD and T N F without serum. After 18 h of incubation, cell lysis was determined by crystal violet staining (see Materials and Methods section) and by the proposed method. One can see from Fig. 3 that the results obtained by both methods were quite similar and in accordance with the literature (Coffman et al., 1988; Mace et al., 1988). We further verified the applicability of the method for the evaluation of cell death in suspension. Isolated rat thymocytes were irradiated and after 7 h of incubation the percentage of dead cells was determined. For comparison cells stained with trypan blue were counted by microscopy. Figure 4 shows that the results were essentially the same and analogous to the radiobiological data (Afanasiev et al., 1988).
specific fluorescent dyes, such as Hoechst 33258, DAPI and EtBr, only EtBr is satisfactory for the automatic scanning of plates. Not only do Hoechst 33258 and D A P I penetrate living cells slowly, but their fluorescence spectra are significantly overlapped by that of the plates (~ .... = 505 and 465 nm, respectively). In this study the exciting wavelength of 300 nm was chosen for the following reasons. First, it is one of the optimum exciting wavelengths for EtBr (Osterman, 1984); second, at this 2% value the emission of plates at 610 nm is negligible (Fig. 1) and, finally, such a wavelength is provided in some automatic readers (for example, Fluoroscan II, Flow). The proposed method has some further advantages over other techniques. First, the preliminary separation of dead and living cells is not obligatory. As stated above this simplifies the procedure and also makes it possible to evaluate both the cytotoxic and cytostatic effects simultaneously. Second, cell debris, formed during long incubations and undetectable by method other than flow cytometry (Deitch, 1987), can be taken into account in this system. Third, the consecutive staining of dead cells and subsequently the whole population permits one to determine the proportion of dead cells in the control samples. Thus an evaluation of absolute cytolysis values becomes possible. Usually such values (the actual value of the test sample minus that of the control sample) are lower than those obtained, for example, by crystal violet staining (Fig. 3). In addition it should be noted that the proposed method is applicable to the analysis of both dividing and quiescent cells as well as plated and suspension cell cultures.
Discussion In some circumstances cytotoxicity cannot be correctly determined by the automatic scanning of plates with cells stained by traditional methods, since vital and supravital dyes differ in their properties. It would be advantageous to use a compound possessing properties of both vital and supravital dyes and some fluorescent dyes exhibit such properties. They are able, on one hand, to enter dead cells and, on the other hand, their emission increases markedly on binding to nucleic acids. However, among widely used nucleic acid-
Acknowledgement We would like to thank V. Shlektarev for technical assistance.
References Afanasiev, V.N., Korol, B.A., Matylevich, N.P., Pechatnikov, V.A., Sorokina, N.I., Umansky, S.R. and Filippovich, l.V. (1988) General regularities and variations in the postirradi-
205 ation death rate of lymphocytes of various animal species and man. Radiobiologiya (U.S.S.R.) 28, 731. Beletsky, I.P., Matyasova, J., Nikonova, L.V., Scalka, M. and Umansky, S.R. (1989) On the role of Ca,Mg-dependent nuclease in the postirradiation degradation of chromatin in lymphoid tissues. Gen. Physiol. Biophys. 8, 381. Coffman, F.D., Green, L.M. and Ware, C.F. (1988) The relationship of receptor occupancy to the kinetics of cell death mediated by tumour necrosis factor. Lymphokine Res. 7, 371. Deitch, A.D. (1987) The effect of cytotoxicity on DNA histograms. Cytometry 8, 339. Duke, R.C., Chervenak, R. and Cohen, J.J. (1983) Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. J. Immunol. 80, 6361. Hacket, R.J., Davis, L.S. and Lipsky, P.P. (1988) Comparative effects of tumour necrosis factor-a and IL-lfl on mitogeninduced T cell activation. J. Immunol. 140, 2639. Jiromsky, V.V., Medvedev, A.I., Schichkov, V.V., Medvedev, B.I., Ivanov, E.G. and Schirogorov, A.A. (1978) Spectrofotometer-densitometer. Pribori Tekhn. Eksp. (U.S.S.R.) 6, 204. Johnson, L.K., Lan, N.C. and Baxter, J.D. (1979) Stimulation and inhibition of cellular function by glucocorticoids, J. Biol. Chem. 254, 7785. Kawakami, M., Watanabe, N., Ogawa, H., Kato, A., Sando,
H., Yamada, N., Murase, T., Takaku, F., Shibata, S. and Oda, T. (1989) Cachectin/TNF kills or inhibits the differentiation of 3T3-L1 cells according to developmental stage. J. Cell. Physiol. 138, 1. Kronke, M., Hensel, G., Schluter, C., Scheurich, P., Schutze, S. and Pfizenmaier, K. (1988) Tumour necrosis factor and lymphotoxin gene expression in human tumour cell lines. Cancer Res. 48, 5417. Lynch, M.P., Nawaz, S. and Gerschenson, L.E. (1986) Evidence for soluble factors regulating cell death and cell proliferation in primary cultures of rabbit endometrial cells grown on collagen. Proc. Natl. Acad. Sci. U.S.A. 83, 4784. Mace, K.F., Ehrke, M.J., Hod, K., Maccubbin, D.L. and Mihich, E. (1988) Role of tumour necrosis factor in macrophage activation and tumoricidal activity. Cancer Res. 48, 5427. Meager, A., Leung, H. and Woolley, J. (1989) Assays for tumour necrosis factor and related cytokines. J. Immunol. Methods 116, 1. Nissen-Meyer, J., Austgulen, R. and Espevik, T. (1987) Comparison of recombinant tumour necrosis factor and the monocyte derived cytotoxic factor involved in monocytemediated cytotoxicity. Cancer Res. 47, 2251. Osterman, L.A. (1984) In: Methods of Protein and Nucleic Acids Research. Springer Verlag, Berlin, p. 150.