Journal of lmmunological Methods, 156 (1992) 47-54 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00

47

JIM 06487

Direct visualisation and quantification of cellular cytotoxicity using two colour fluorescence Bart-Jan Kroesen, G. Mesander, J.G. ter Haar, T.H. The and L. de Leij Department of Clinical Immunology, Internal Medicine, University Hospital Groningen, Oostersinge159, 9713 EZ Groningen, Netherlands (Received 17 February 1992, revised received 8 May 1992, accepted 5 June 1992)

A fluorescence method is described for the evaluation of cell death induced by cellular cytolytic activity. A green fluorescent membrane dye, D275, was used to label various target cell lines and propidium iodide (PI) uptake was used to assay cell death. Natural killer (NK), lymphokine activated killer (LAK) as well as targeted T cell mediated cellular cytotoxicity were quantified using the fluorescence method and compared to results obtained with the 51chromium (51Cr) release assay. A good correlation was found after an assay period of 4-8 h indicating that the fluorescence method is a reliable alternative to the 51Cr release assay. Key words: Membrane fluorescence; Cellular cytotoxicity; Natural killer cell; Lymphokine activated killer cell; Activated T cell

Introduction

Cell-mediated or cellular cytotoxicity is a process in which target cells are recognized and subsequently killed by effector cells (Ullberg et al., 1981; Menzer et al., 1987). The standard procedure to quantify cellular cytotoxicity is the 51Cr release assay. This procedure requires a

Correspondence to: B.J. Kroesen, Department of Clinical Immunology, Internal Medicine, University Hospital Groningen, Oostersingel 59, 9713 EZ Groningen, Netherlands. Abbreviations: 5~Cr, sodium-Slchromate; 3H-thym, [3H] thymidine; BCEF, bis-carboxyethyl-carboxyfluorescein; H33342, Hoechst dye no. 33342; LDH, lactate dehydrogenase; PI, propidium iodide; D275, 3,3'-dioctadecyloxacarbocyanine perchlorate (DIOC18); NK, natural killer cell; LAK, lymphokine activated killer cell; TcR, T cell receptor; IL-2, interleukin-2; DMSO, dimethyl sulfoxide; PBS, phosphatebuffered saline; PBL, peripheral blood lymphocytes; E / T ratio, effector-to-target cell ratio; FcR, Fc receptor; Mab, monoclonal antibody.

pre-labelling of target cells with 51Cr after which they are incubated with effector cells. The released 51Cr label is subsequently measured and gives an indirect measure of target cell death (Brunner et al., 1968). Although the reliability of this method is well established, it has some disadvantages. A laboratory approved for working with radioactive compounds is needed, and the relatively high cost of 51Cr has to be considered. Due to its comparatively short half-life (28 days), new supplies of 51Cr have to be ordered regularly which makes the procedure expensive if cytotoxicity assays are performed on a non-routine basis. An additional disadvantage is that 51Cr may bind poorly to or show high spontaneous release from some target cell lines. Other methods for the quantification of cytotoxicity have been described and include measurement of released compounds such as incorporated [3H]thymidine, fluorescent markers (e.g., BCEF, europium and H33342) or enzymes such as LDH (Keilholz et al., 1990;

48 Kolber et al., 1988; Blomberg et al., 1986a, b; Volgmann et al., 1989; Brenan and Parish, 1988; Szekeres et al., 1981; Korzeniewski et al., 1983). Here a method is described to visualize and quantify cell death directly using specific fluorescent labels and fluorescence microscopy. In this method, target cells are stained using a green fluorescent membrane dye, 3,3'-dioctadecyloxacarbocyanine perchlorate (DIOC18 or D275), which can be incorporated into the cell membrane by its two C18 alkyl chains (Badley et al., 1973). As a result, the stained target ceils become distinguishable in the assay from the subsequently added unlabelled effector cells. After the incubation period in the cytotoxicity assay, propidium iodide (PI) is added which only penetrates the dead cells and specifically stains their nuclei a fluorescent red. In this way, dead target cells can be recognized by fluorescence microscopy as g r e e n / r e d double-fluorescent cells, whereas living target cells are green mono-fluorescent. Living effector cells are invisible while dead effector cells are red mono-fluorescent and thus distinguishable from dead target cells. In this report the method is compared with the standard 5~Cr release assay using various e f f e c t o r / t a r g e t combinations for different types of cytotoxicity.

Materials and methods

Cell lines and cell culture NK and LAK activity was assayed using the NK sensitive human erythroblastoid cell line K562, and the NK resistant, LAK sensitive human B lymphoblastoid cell line Daudi. Targeted T cell cytotoxicity was assayed by reverse ADCC using the FcR positive mouse mastocytoma derived cell line P815 (Ralph et al., 1976). All cell lines as well as activated peripheral blood cells (see below) were grown in culture medium consisting of Hepes buffered RPMI 1640 (Gibco Europe, Breda, Netherlands) supplemented with 14% heat inactivated fetal calf serum, 2 mM glutamine, 60 i z g / m l gentamicin (Schering, Kenilworth, USA), 0.05 mM ~-mercaptoethanol and 1 mM sodium-pyruvate. Cells were grown in a CO 2 incubator (5% CO z) in a humidified atmosphere at 37°C.

Antibodies For activation of human peripheral blood lymphocytes (PBL),"the monoclonal antibody (Mab) WT32, recognizing the e chain of the human CD3 complex (Tax et al., 1981) was used. Activation, as well as targeting the lytic potential of rat peripheral blood lymphocytes derived T cells, was achieved with Mab R73, which recognizes a constant determinant on the TCR of rat T ceils (Hfinig et al., 1989). Isolation and activation of effector cells Human effector cells were isolated from heparinized blood obtained from healthy volunteers. Density centrifugation of blood diluted 1/1 with phosphate-buffered saline (PBS) was done on Lymphoprep gradients (Nycomed Pharma AS, Oslo, Norway; density at 20°C: 1.076) at 2400 rpm for 20 min. After collecting the cells from the interface, they were washed twice in RPMI 1640 containing 60 Ixg/ml gentamicin and resuspended in culture medium. Rat effector cells were isolated from heparinized blood obtained from male (AO*BN)F1 rats by density centrifugation of blood diluted 1/1 with PBS on Percoll (Pharmacia, Upsala, Sweden) density gradients, set to a final density of 1.085 g / m l with PBS, at 2400 rpm for 20 min. The cells at the interface ring were harvested, washed as above for the human effector cells and resuspended in culture medium. All effector cells were activated for 5 days using T cell activating antibodies (WT32 or R73) and low doses rIL-2 (Eurocetus, Amsterdam, The Netherlands). For activation of human effector cells, isolated PBL were initially incubated in culture medium supplemented with 5% (v/v) WT32 supernatant. After 3 days the cells were washed once, resuspended in fresh culture medium supplemented with 30 Cetus U IL-2/ml, and incubated for 2 additional days. Activation of rat effector cells was performed as described for human effector cells except that R73 Mab (5% v / v culture supernatant) was used as a T cell activating antibody and 50 U I L - 2 / m l was used in the second incubation period. Although these activation protocols are designed to preferentially activate T cells (Van Wauwe et al., 1980) some LAK activity is also induced (Patel et al., 1987).

49 Therefore these effector cell populations can be used to assay the potential of both activated T cells and LAK cells.

Target cell labelling D275 label: D275 (Molecular Probes, Eugene, USA) has a fluorescein-like spectrum (excitation: 484 nm; emission: 507 nm), a MW of 880 daltons, and is used in the experiments from a stock solution of 2.5 m g / m l prepared in DMSO (Merck, Darmstadt, Germany). D275 labelling: 5 x 106 target cells were labelled overnight by incubation in culture medium supplemented with D275 at a final concentration of 10 /zg/ml. The final concentration of DMSO during this labelling was approximately 0.4% which did not influence cell viability. After labelling, cells were washed once in culture medium to remove free label and used for the cytotoxicity assay as described below. 51Cr labelling: 5 x 10 6 target cells were washed once in PBS and labelled by incubation in 100 tzCi sodium-5~chromate (Amersham, Buckinghamshire, UK) in 100/zl PBS for 1 h at 37°C, 5% CO 2. After labelling, cells were washed three times with culture medium to remove free label and used for the cytotoxicity assay as described below.

Flow cytometric analysis of D275 labelled cells The stability of the D275 label in labelled target ceils was examined by flow cytometric analysis. For this purpose, a 1 : 1 mixture of 5 x 106 D275 labelled and unlabelled K562 target cells was incubated for various periods of time in culture medium at 37°C, 5%CO 2. After the incubation, samples at each time point were analysed by flow cytometry using a FACS 440 (Becton Dickinson, Mountainview CA, USA). Fluorescence intensity was recorded in arbitrary units.

Quantification of cytotoxicity By fluorescence microscopy: for cytotoxicity, 1 x 10 4 D275 labelled target cells were incubated with effector cells in effector to target cell ratios ( E / T ratios) of 5 and 50 in a final volume of 1 ml in culture medium supplemented with 20 U IL2/ml. After incubation for various periods of time at 37°C, and 5% CO 2 in culture tubes (Greiner no. 120180), the ceils were spun down

and resuspended in 50/zl PBS containing 5/.~g/ml PI. Immediately thereafter cell death was quantified by fluorescence microscopy with a 495 nm filter permitting assessment of both green and red fluorescence. Each sample was counted twice in random order and each time 100 green fluorescent cells per sample were scored. Cell death was determined as the percentage of g r e e n / r e d double fluorescent ceils per 200 green fluorescent cells counted. By 51Cr-release: after labelling target ceils with SaCr, 2 x 103 target cells were incubated with effector cells in E / T ratios of 5 and 50 in a final volume of 200 /zl culture medium supplemented with 20 U I L - 2 / m l and incubated for various periods of time at 37°C, 5% CO 2 in 96-well microtiterplates (Greiner no. 650180). After incubation, the plates were centrifuged at 200 × g for 5 min and 100 ~1 samples were collected and counted in a gamma-counter. Cell death was calculated according to the formula: cell death = (experimentalrelease - spontaneous release) /(maximal release - spontaneous release) X 100% Spontaneous release was determined as the number of counts released after incubation of target cells in medium alone, whereas maximal release was determined as the number of counts released from the target cells after incubation in medium supplemented with 2.5% Triton X-100.

Effector cell targeting After activation, rat effector cells were incubated with R73 (5% culture supernatant v / v ) for 30 min at 0°C. Subsequently the cells were washed once in culture medium to remove unbound R73, resuspended in culture medium supplemented with 50 Cetus U I L - 2 / m l and used for cytotoxicity against the FcR positive target cell line P815 as described for cytotoxicity against K562 and Daudi target cells.

Results

Labelling of target cells with D275 Pilot experiments were performed to examine the properties of the D275 label, after cell la-

50

belling, in relation to time. For this purpose K562 target cells were labelled overnight with D275, mixed 1:1 with unlabelled K562 cells and incubated in culture medium for 2, 4, 8 and 24 h at 37°C, 5% CO 2. D275 remained located in the cell membrane of the labelled cells and brightly fluorescent for at least 24 h as depicted in Figs. la-le. The observed decrease in fluorescence intensity of the D275 labelled cells with time can be explained by dilution as a result of cell division since the label is passed on to the daughter ceils. Any take up of possibly shed label by unlabelled cells from D275 labelled cells remains well within the limits necessary for the discrimination of labelled from unlabelled cells. No D275 label associated toxicity, as measured by a trypan blue uptake assay, was observed after target cell labelling (data not shown).

Labelling of target cells with D275 in itself does not alter the susceptibility of these target cells to lytic attach by effector cells, i.e., N K / L A K activity. This was shown in a 51Cr release assay in which cytotoxicity against D275 labelled K 5 6 2 / Daudi target cells was compared to cytotoxicity against the same non-fluorescent target cells (Figs. 2a and 2b).

Quantification of cytotoxicity To compare susceptibility to cytotoxicity as measured by fluorescence with that measured by 51Cr release, target cells were labelled with D275 or 51Cr and incubated with unactivated or activated effector cells for different periods of time (Figs. 3 and 4). To avoid inter-experimental variation, both types of cytotoxicity assays were done simultaneously using the same batch of effector

1

1

c

d ° ~

t~

fluorescence inumaity Fig. 1. Stability of D275 in labelled cells. K562 cells were labelled overnight with D275. After washing, labelled cells were mixed 1 : 1 with non-fluorescent K562 cells and incubated for various times. Using flow cytometry, the fluorescence intensity of the cells was recorded against the size of the cell population in arbitrary units after 0 (a), 2 (b), 4 (c), 8 (d) and 24 (e) h of incubation at 37°C, 5%CO 2.

51 % cell death (51Cr)

% cell death (51Cr)

80

80

70

70

60

60

50

50

E3

©

40

40

30

30

20

20

Q 10

/x

10

0 0

I

I

I

I

I

I

I

10

20

30

40

50

60

70

0 80

0

10

% cell death (51Cr÷D275)

20

30

40

50

60

70

80

% cell death (51Cr+D275) b

I

Fig. 2. Influence of target cell labelling with D275 on susceptibility to NK and LAK activity. D275 labelled or non-fluorescent K562 (a) or Daudi (b) cells were radiolabelled with 5:Cr and used as targets for NK or LAK mediated cytotoxicity. Effector cells were added in effector to target ratios of 5 (closed symbols) and 50 (open symbols). After 2 (o and ©), 4 ( • and zx) and 8 ( • and []) h, cytotoxicity against the target cells was measured by assessing 51Cr release. Percentage 5:Cr release from cells labelled with 51Cr only (51Cr) is plotted against that released from cells labelled with both D275 and 51Cr (D275 + 51Cr).

cells, split just prior to the experiment. Operators measuring cytotoxicity by fluorescence were unaware of the results of the 51Cr release. Determination of cytotoxicity was done by counting the samples twice in random order but the variation in the number of dead cells counted never exceeded 5%. Fig. 3a shows normal peripheral blood NK activity as measured against K562 target cells whereas Fig. 3b, shows the LAK activity as assayed against Daudi target cells. A good correlation between quantification of cytolytic activity as measured by 5~Cr release and that determined by PI uptake is seen. This is true between 4 and 8 h, whereas after 2 h cytotoxicity, hardly any released 51Cr is measured although some target cells already appear to be killed as measured by PI uptake. The reproducibility of the method is shown in Table I which summarises three successive comparative cytotoxicity experiments using effector cells from two different donors. The results from another form of cellular cytotoxicity, in which activated cytotoxic T cells are

focussed towards target cells (T cell targeting) are shown in Fig. 4. For this kind of cytotoxicity, rat peripheral blood lymphocytes were activated and used as effector cells. Focussing was done by means of the Mab R73, recognizing the T cell receptor on rat T cells, which forms a functional bridge between the effector cells and the FcyRIII positive target cell line P815. For this kind of cytotoxicity, a good correlation is seen between the quantification of cytolytic activity as determined by 5:Cr release and that measured by PI uptake (Fig. 4).

Discussion

The purpose of the present study was to test the reliability of an assay based on fluorescence for the measurement of cellular cytolytic activity. Pre-labelling target cells with a green fluorescent membrane dye was done in order to be able to discriminate easily between dead target cells and dead non-target cells. In particular when high

52 cell death (%)

cell death (%)

601 A

180

cell death (%)

50 50

4O 30

f

/

40 20

10

10

0~0

5

50

0

E / T ratio

5

30

0 50

E / T ratio

20

f /ffff-

10 cell death (%) 60 r

c e l l death (%)

,otA

~

50

4O

-20

10

10

5

50 E / T ratio

2 0

0

5

0 50

5 E/T ratio

50

Fig. 4. Targeted cellular cytotoxicity against P815 target cells. Activated rat PBL were targeted (*) with R73 Moab, or not (e), and incubated with P815 target cells in different effectorto-target ratios. Target cells were either pre labelled w i t h D275 ( ) or 51Cr ( . . . . . . ). After 4 h incubation of effector cells with target cells, cell death was quantified by assessment of PI uptake or 5~Cr release.

E / T ratio

Fig. 3. a: NK activity against 5~Cr labelled K562 target cell as determined by 51Cr release (a,4), and D275 labelled K562 target cells as determined by fluorescence microscopy (aB). D275 and 51Cr labelled K562 target cells were incubated with freshly isolated PBL in effector to target ratios of 5 and 50. In the control ( E / T = 0), no effector cells were added. After 2 (e), 4 ( + ) and 8 (*) h, cell death was quantified, b: LAK activity against 51Cr labelled Daudi target cells as determined by 51Cr release (bA), and D275 labelled Daudi target cells as determined by fluorescence microscopy (bB). D275 and 51Cr labelled Daudi target cells were incubated with activated PBL in effector to target ratios of 5 and 50. In the control ( E / T = 0), no effector cells were added. After 2 (e), 4 ( + ) and 8 (*) h, cell death was quantified.

E/T

G

40

20

0

/'""

60

ratios are used this might prevent overe s t i m a t i o n o f c y t o t o x i c i t y s u c h as w h e n m e t h o d s are used based on MTT staining for measurement of viability (Mosmann, 1983) or PI uptake a l o n e f o r m e a s u r e m e n t o f cell d e a t h . Our strategy has been to pre-label target cells with a hydrophobic, membrane intercalating dye

rather than protein reactive compounds F I T C . I n t h i s way, t h e t a r g e t c e l l s w e r e c h a n g e d as p o s s i b l e a n d w e r e n o t e x p o s e d doses of radioactivity. Another advantage l a b e l l i n g p r o c e d u r e is t h e p o s s i b i l i t y o f

s u c h as as l i t t l e to high of this further

TABLE I COMPARISON OF CYTOTOXICITY (% CELL DEATH) AGAINST K562 CELLS, MEASURED BY 51Cr RELEASE AND FLUORESCENCE MICROSCOPY Successive cytotoxicity experiments with K562 target cells and effector cells ( E / T ratio = 50) from two different donors. Cytotoxicity was measured by released 51Cr from target cells (51Cr) or by fluorescence microscopy (FM) in which the percentage of dead target cells per 200 target cells was counted. Cytotoxicity stopped after

Exp. 1 51Cr FM

Exp, 2 51Cr FM

Exp. 3 51Cr FM

2h 4h 8h

6 18 25

3 10 15

2 17 34

10 25 32

5 12 24

11 20 41

53 manipulation of the target cells such as labelling with ta5IdUd for the m e a s u r e m e n t of apoptotic processes induced by effector cells (Zychlinsky et al., 1992). Fig. 2 indicates that susceptibility to N K / L A K activity is not altered when target cells are labelled with D275. Furthermore, after labelling, the label is anchored firmly in the cell membrane, making longer experiments possible without the loss of fluorescence or uptake of shed label by unlabelled cells (Fig. 1). The results in Figs. 2, 3 and 4 indicate a good correlation between quantification of cytotoxicity as measured by 5lCr release from, and that determined by PI uptake in, dead target cells in NK, L A K and targeted T cell mediated cytotoxicity. Fig. 3 shows that the induction of a percentage specific cell lysis as determined indirectly by a 4 or 8 h 5iCr release assay in fact correlates with the same percentage cell death as determined directly by uptake of PI. When the incubation time is shorter, for example 2 h, a 5~Cr release assay appears to give an underestimation of cell death. Apparently PI is taken up more quickly in dead cells than 5~Cr is released from these cells. Different incubation times for cytotoxicity were examined. The correlation between quantification by fluorescence microscopy and 51Cr release was optimal for assay times between 4 and 8 h. In cytotoxicity assays run for more than 8 h, it was difficult to quantify cell death using the fluorescence method due to the fact that a n u m b e r of target cells began to disintegrate. Some inter-experimental variation was found between experiments in which effector cells from different donors were used (Table I), however inter-assay variation between the 51Cr release and the fluorescence method for quantification of cytotoxicity within one experiment was limited. Although the results as shown here were reproducable, statistical reliability of the quantification could be upgraded by counting larger numbers of target cells. Semi-automated fluorescence image analysers capable of counting and interpreting large numbers of cells might prove useful in this respect. To date, we have not been successful in analysing cytotoxicity by the D 2 7 5 / P I two colour fluorescence method using flow cytometry. This was mainly due to conjugate formation between effector and target cells, induced during the process of

cytotoxicity (Menzer et al., 1987; Ullberg et al., 1981). These conjugates, consisting of both dead and live target as well as effector cells, made interpretation of the results impossible, especially when high effector to target ratios and antibody targeted T cells were used in the assay. Besides disturbance of the flow through, one of the main practical problems in this respect was adhesion of dead effector ceils to living target cells which resulted in an overestimation of dead ( g r e e n / r e d double fluorescent) target cells. Quantification by flow cytometry of conjugates per se, as formed during cytotoxicity has been reported (Radcliff et al., 1991). This, however, will only give a rough and indirect indication of cellular cytotoxicity. We have shown here that quantification of cellular cytotoxicity based on two colour fluorescence as described in this report, is a direct, fast and inexpensive alternative to the standard 51Cr release assay.

Acknowledgements The authors thank W. Tax and T. Hiinig for kindly providing the monoclonal antibodies WT32 and R73 respectively, and B. Schilizzi for carefully reading the manuscript. This work was supported by a grant from the Dutch Cancer Society (Koningin Wilhelmina Fonds, 89-07).

References Badley, R.A., Martin, W.G. and Schneider, H. (1973) Dynamic behaviour of fluorescent probes in lipid bilayer model membranes. Biochemistry 12, 268. Blomberg, K., Granberg, C., Hemmil~i, I. and L6vgren, T. (1986a) Europium-labelled target cells in an assay of killer cell activity. I. A novel non-radioactive method based on time resolved fluorescence. J. Immunol. Methods 86, 225. Blomberg, K., Granberg, C., Hemmil~i, I. and L6vgren, T. (1986b) Europium-labelled target cells in an assay of killer cell cryopreserved target cells pre-labelled with the fluorescentactivity. II. Significance and specificity of the method. J. Immunol. Methods 92, 117. Brenan, M. and Parish, C.R. (1988) Automated fluorometric assay for T cell cytotoxicity.J. Immunol. Methods 112, 121. Brunner, K.T., Mauel, J. Cerottini, J.C. and Chapuis, B. (1968) Quantitative assay of the cytolytic action of immune

54 lymphoid cells on 51Cr-labelled allogeneic target cells in vitro: inhibition by isoantibody and by drugs. Immunology 14, 18. Hiinig, T., Wallny, H.J., Hartley, J.K., Lawetzky, A. and Tiefenthaler, G. (1989) A monoclonal antibody to a constant determinant of the rat T cell antigen receptor that induces T cell activation. J. Exp. Med. 169, 73. Keilholz, U., Dummer, R., Welters, H., Brado, B., Galm, F., Matheiowetz, P. and Hunstein, W. (1990) A modified cytotoxicity assay with high sensitivity. Scand. J. Clin. Lab. Invest. 50, 879. Kolber, M.A., Quinones, R.R., Gress, R.E. and Henkart, P.A. (1988) Measurement of cytotoxicity by target cell release and retention of the fluorescent dye bis-carboxyethylcarboxyfluorescein (BCEF). J. lmmunol. Methods 108, 255. Korzeniewski, C. and Callewaert, D.M. (1983) An enzyme-release assay for natural cytotoxicity. J. lmmunol. Methods 64, 313. Menzer, S.J., Smith, B.R., Barbosa, S.A., Crimmins, M.A.V., Herrmann, S.H. and Burakoff, S.J. (1987) CTL adhesion and antigen recognition are discrete steps in the human CTL target cell interaction. J. Immunol. 138, 1325. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. lmmunol. Methods 65, 55. Patel, S.S., Thiele, D.L. and Lipsky, P.E. (1987) Major histocompatibility complex-unrestricted cytolytic activity of human T cells. J. Immunol. 139, 3886.

Radcliff, G., Waite, R., LeFevre, J., Poulik, M.D. and Callewaert, D.M. (1991) Quantification of effector/target conjugation involving natural killer (NK) or lymphokine activated killer (LAK) cells by two-color cytometry. J. Immunol. Methods 139, 281. Ralph, P., Moore, M.A.S. and Nilsson, K. (1976) Lysozyme synthesis by established human and murine histiocytic lymphoma cell lines. J. Exp. Med. 143. Szekeres, I., Pasca, A.S. and Peytsik, B. (1981) Measurement of lymphocyte cytotoxicity by assessing endogeneous alkaline phosphatase activity of the target cells. J. Immunol. Methods 40, 151. Tax, W.J., Leeuwenberg, H.F.M., Willems, H.M., Capel, P.J.A. and Koen, R.A.P. (1981) In: A. Bernard, L. Boumsell, J. Dauset, C. Milstein and S.F. Schlossman (Eds.), Leukocyte Typing. Springer-Verlag, Berlin, p. 721. UIIberg, M. and Jondal, M. (1981) Recycling and target binding capacity of human natural killer cells. J. Exp. Methods 153, 615. Van Wauwe, J.P., De Mey, J.R. and Goossens, J.G. (1980) OKT3: A monoclonal anti-human T lymphocyte antibody with potent mitogenic properties. J. Immunol. 124, 2708. Volgmann, T. Klein-Struckmeier, A. and Mohr, H. (1989) A fluorescence-based assay for quantification of lymphokineactivated killer cell activity. J. Immunol. Methods 119, 45. Zychlinsky, A., Zheng, L.M., Liu, C., Young, J.D. (1992) Cytolytic lymphocytes induce both apoptosis and necrosis in target cells. J. lmmunol. 146, 393.

Direct visualisation and quantification of cellular cytotoxicity using two colour flourescence.

A fluorescence method is described for the evaluation of cell death induced by cellular cytolytic activity. A green fluorescent membrane dye, D275, wa...
559KB Sizes 0 Downloads 0 Views