- Email: [email protected]
[31] Quantitative cytotoxic assay using a coulter counter
[31]
CYTOTOXIC ASSAY WITH COULTER COUNTER
481
[31] Quantitative Cytotoxic Assay Using a Coulter Counter By Y O S H I T A K A
NAGAI, MACHIKO IWAMOTO, and
TOSHIHARU MATSUMURA
Cytotoxicity has been assayed either by determining the number of cells that become unable to exclude a dye, such as trypan blue, or by determining the amount of radioisotope released from preradiolabeled cells. The dye exclusion method is tedious and time consuming, but it is useful for a few samples. An unavoidable delay when many samples are handled causes an increase in the number of stained cells. Moreover, the presence of cell aggregates or erythrocytes causes interference. The radioisotope method is suitable to handle many samples, but it requires expensive instruments and reagents. Sometimes, spontaneous release of radioisotope complicates the analysis. It has been known that damaged cells are more susceptible to protease treatment than intact cells. To quantitate complement-mediated immune cytolysis of lymphosarcoma cells and mouse spleen cells, Stewart and Goldstein ~ combined the procedure of protease digestion with that of electronic counting. In this method, cells are first treated with a protease to completely digest damaged cells and subsequently with a detergent to lyse intact cells as well as erythrocytes. The number of residual nuclei from intact cells is determined electronically. We modified and extended the method of Stewart and Goldstein. This assay 2 is rapid and reproducible and gives results comparable with those given by a dye-exclusion method. As our objective was to develop a rapid and quantitative method for the determination of antibody-mediated cytotoxicity, we describe examples using thymocytes as target cells. This cytotoxicity assay, when appropriately modified, is applicable to the determination of viability of tissue culture cells. Therefore, examples using L-929 mouse fibroblasts and FM3A mouse mammary carcinoma cells are also described. Application to Antibody-Mediated Cytotoxicity
Reagents The medium used in all procedures is Eagle's MEM, adjusted to pH 7.2 with 7.5% NaHCO3. Pronase E (Kakenkagaku Company, Tokyo, C. C. Stewart and S. Goldstein, J. Lab. Clin. Med. 84, 425 (1974). 2 M. I w a m o t o and Y. Nagai, Jpn. J. Exp. Med. 51, 109 (1981).
METHODS IN ENZYMOLOGY,VOL. 132
Copyright © 1986by Academic Press, Inc. All rightsof reproductionin any form reserved.
482
ASSAYS FOR CYTOTOXIC ACTIVITY
[31]
Japan) 3 is dissolved in phosphate-buffered saline (PBS), 3a pH 7.2, at a concentration of 3 mg/ml and stored at - 2 0 ° in 2- to 3-ml aliquots. Pronase (purchased from Calbiochem) can be used as well as Pronase E. The frozen stock solution is thawed just before use and is filtered through a 0.45-/xm millipore filter for removal of interfering precipitates. Isoton II and Zap-Oglobin are purchased from Coulter Diagnostics (Hialeah, FL). Zap-Oglobin is a hemolysing agent for diagnostic use, which lyses erythrocytes and the membranes of nucleated cells to liberate the nuclei of leukocytes for counting. Trypan blue is dissolved in PBS at a concentration of 0.2%. Lyophilized guinea pig serum, dissolved in doubly distilled water and diluted 1 : 4 with medium, is used as complement after absorption with agar. 4 Although any medium appropriate for the culture of target cells may be used, addition of 10 mM HEPES and 1% BSA will prevent cell damage.
Target Cells Thymus of 5- to 8-week-old male C3H/He mouse is removed and teased apart with sharp forceps in medium. The cell suspension is pipetted repeatedly and then filtered through a stainless steel screen (200 mesh). For further details, see this series, Vol. 108 [6]. To obtain a 100% dead cell suspension, a portion of the thymocyte suspension is incubated at 56° for 30 min. The untreated portion is used as 100% viable cells. The two cell suspensions are diluted with medium to 5 × 106 ceils/ml, equal volumes of the two suspensions are mixed and used as 50% dead cells. Viability of these suspensions, measured with trypan blue dye exclusion, gave a value 2-7% dead cells for viable cells, 45-53% dead cells for 50% dead cells, and 100% dead cells for the 100% dead cell preparation.
Coulter Counter Setting In our experiments, a Model ZBI Coulter counter was used with a 100/~m orifice aperture tube. The common settings of the counter were as follows: manometer, 0.5 ml; matching switch, 20 K; gain trim, 0. The threshold settings for thymocyte nuclei were A (l/amplification), 1/2; I (1/aperture current), 0.354; TL (lower threshold), 15; To (upper threshold), 60 (nuclear threshold). The settings for intact thymocytes were A, 1; I, 1/2; TL, 15; Tu, 60 (cellular threshold). The K value of our Coulter counter was measured as 11.5, so that the volume difference per each threshold was 2.04/zm 3 for nuclear threshold and 5.75 p . m 3 for cellular threshold. 3 T h e n a m e Pronase E was c h a n g e d to Actinase E recently. 3a Abbreviations: PBS, phosphate-buffered saline; BSA, bovine s e r u m albumin. 4 A. C o h e n and M. Schlesinger, Transplantation 10, 130 (1970).
[31]
C Y T O T O X I C ASSAY W I T H C O U L T E R C O U N T E R
483
Analysis of Volume Frequency Distribution In order to obtain a precise counting of the target cells and an effective rejection of debris, the most important adjustment is the threshold setting. The volume distribution pattern of thymocyte nuclei is first examined to determine the threshold setting. Thymocyte suspensions diluted with Isoton II with or without Zap-Oglobin (three drops/10 ml of counting solution) are analyzed for volume frequency distribution. Figure 1A illustrates the volume distribution of a thymocyte suspension treated with Zap-Oglobin, and it is obtained from counting the number of particles in each threshold at a setting ofA = 1/2 and I = 0.354, which corresponds to 2.04 /xm3 per each threshold. In Fig. I A, two populations of particles are seen. The first distribution, showing a marked decrease from a threshold of 1 to 10, is due to debris. The second distribution, giving a peak at a threshold of about 30, is due to thymocyte nuclei. The mean volume of the nuclei was calculated to be 67 / z m 3. According to this distribution pattern, the particles which are counted between threshold 15 and threshold 60 are measured as thymocyte nuclei. On the other hand, the thymocyte suspension without Zap-Oglobin shows a similar distribution pattern at a setting A = 1 and I = 1/2, and the mean volume is 178/zm 3 (Fig. IB). The volume distribution pattern of erythrocytes (mean volume 77 tzm3) overlaps with that of thymocyte nuclei; erythrocytes, however, are completely solubilized by addition of Zap-Oglobin (Table I). Therefore, contaminating erythrocytes do not interfere in the cytotoxicity assay.
Recovery of Cell Counts after Treatment with Zap-Oglobin and Pronase E Addition of Zap-Oglobin after incubation with Pronase E results in the liberation of nuclei from the remaining viable cells. The effects of ZapOglobin and protease treatment on viable cells and dead cells are shown in Table I. Cells (5 x 105) of each suspension in 0.3 ml of medium containing 1% BSA are incubated with 0.3 ml of PBS or 0.3 ml of 3 mg/ml of Pronase E at 37°. After 30 min, 0.5 ml of the mixture is added to a counting vial containing 10 ml of Isoton II and is counted at cellular threshold (values under column headings 1 and 3, in Table I). Then, three drops (0.1 ml) of Zap-Oglobin are added to the vial and the sample is counted once more at nuclear threshold (values under column headings 2 and 4, in Table I). For the calculation of cell recovery after treatment with Zap-Oglobin and Pronase E, the complete recovery is set at the counts with ZapOglobin but without Pronase E treatment.
484
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Threshold FIG. |. (A) Volume frequency distribution patterns of thymocyte nuclei. Sample treated with Isoton I1 and Zap-Oglobin. The mean volume of thymocyte nuclei (-~) is 67 /~m3. A = 0.5, I = 0.354, 2.04/~m 3 per each threshold. (B) Volume frequency distribution patterns of thymocytes (solid line), sample treated with lsoton 11 only, and thymocyte nuclei (broken line). The mean volume of thymocytes (,~) is 178/~m 3 and that of nuclei (,~) is 67/~m 3. A = !, ! = 0.5, 5.75/~m 3 per each threshold.
[31]
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ASSAYS FOR CYTOTOXIC ACTIVITY
TABLE II DOSE RESPONSE OF PRONASE E TREATMENT
Percentage decrease from Pronase E-free control Pronase E concentration (mg/ml)
Viable cells
50% Dead cells
100% Dead cells
0 1 2 3
0 0 1.0 0.4
0 39.5 43.2 48.5
0 89.5 88.5 96.1
T r y p a n blue a
5.0
50.0
100
a Percentageof dead cells estimated by the trypan blue dye exclusion method without Pronase E treatment.
In the case of a viable cell suspension, there is no difference in the counts for cells and nuclei, suggesting that the effect of protease is negligible (the percentage viability by dye exclusion is 97.2% and the percentage recovery with Pronase-Zap treatment is 94.7%). However, protease decreases the count of dead cells by about 80%. Addition of Zap-Oglobin causes the complete destruction of dead cells, yielding background level counts. When erythrocytes are present together with the target cells, they are completely solubilized with Zap-Oglobin, and so have no effect on the count of the target cells.
Effect of the Amount of Pronase E and of the Incubation Time The dose dependence of the effect of Pronase E on viable and dead cells is shown in Table II. Satisfactory results are obtained with Pronase E at 3 mg/ml. With this amount the observed lysis is 0.4% for viable cells, 48.5% for 50% dead cells, and 96.1% for 100% dead cells. Only partial digestion is observed at lower protease concentrations, and 5 mg/ml Pronase E has the same effect as 3 mg/ml Pronase E. The percentage of cells digested at 37° as a function of time does not change appreciably. After incubation at 37° for 30 min, only 5% of the viable cells are lost, but the percentage lysis of 100% dead cells is 98.5%, and that of 50% dead cells is 52.4%. Similar results are obtained after incubation for 60 min.
[31]
487
CYTOTOXIC ASSAY WITH COULTER COUNTER TABLE III REPRODUCIBILITY OF PRONASE E AND ZAP-OGLOBIN PROCEDURE Viable cells
Well number 1 2 3 4 5 Average SD c
Count displayed" 16,286 16,588 16,758 16,399 16,524
16,305 16,673 16,782 16,490 16,621
50% Dead cells
Number of cells/mlb 0 0 -4) 3.40 3.48 3.52 3.43 3.45 3.46 0.05
Count displayed 7,593 7,711 7,875 7,991 7,630
7,407 7,823 8,035 7,892 7,536
Number of cells/ml (10 4) 1.50 1.55 1.59 1.59 1.51 1.55 0.04
100% Dead cells
Count displayed 128 160 132 129 125
106 128 112 135 160
Number of cells/ml (10 -4) 0.02 0.03 0.02 0.03 0.03 0.03 0.01
a Number of particles in 0.5 ml of counting solution at a setting of A = 0.5, 1 = 0.354, TL = 15, Tu = 60.
b Sum of two displayed counts indicating cells/1 ml of counting solution. If the displayed count is over 10,000, the value is corrected by the available correction table of the Coulter counter. c Standard deviation.
Reproducibility of the Method To test the reproducibility of this method, five samples of each cell suspension are treated with 3 mg/ml of Pronase E at 37 ° for 30 min, then counted with Zap-Oglobin at the nuclear threshold. The results listed in Table III demonstrate the high reproducibility of this method. Furthermore, the counts of each cell suspension are stable for at least 3 hr, z which gives adequate time for measuring many samples at one time.
Assay Procedure of Antibody-Mediated Cytotoxicity Target cells (5 × 105 t h y m o c y t e s in 0. I ml of medium) are added to a plastic tube containing 0.1 ml of diluted antiserum and 0.1 ml of complement. Normal serum is present instead of antiserum in the controls. Each reaction mixture is incubated for 1 hr at 37 ° in a humidified atmosphere of 5% CO2-95% air. After incubation, 0.3 ml of Pronase E solution is added to each tube and the mixtures are further incubated for 30 min. At the end of the incubation, 0.5 ml of the final mixture from each tube is transferred to a counting vial containing 10 ml of Isoton II and three drops (0.1 ml) of Zap-Oglobin. After mixing, the nuclei in the solution are counted in a Coulter c o u n t e r at nuclear threshold. Counts displayed in the counter window are the n u m b e r of particles in 0.5 ml of counting solution. Counts/ 1 ml of the solution are calculated as the sum of the counts determined by
488
[31]
ASSAYS FOR CYTOTOXIC ACTIVITY
5 x 106/ml thymocyte 0.1 ml diluted antiserum 0.1 ml complement x 5 0.1 ml /
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0. 2% trypan blue 0.1ml
~'~ 3mg/mlPronaseE 0.3ml 370C, 30 min in 5% CO2 in air
Hemocytometer counting
0.5 ml sampling
~Isolon II 10 ml p Oglobm 0.1 ml
Dye Exclusion Method Coulter Counter counting Coulter Counter Method FIG. 2. Procedures for the dye-exclusion and Coulter counter methods.
counting the same sample twice. Results are expressed as follows: Percentage lysis (percentage decrease) = (I
counts in sample~ - counts in control/ 100
All tests are performed at least in triplicate. The procedures are summarized in Fig. 2, together with the dye-exclusion method. When a titration of anti-mouse brain-associated theta (0) serum with C3H/He mouse thymocyte is carried out by these two procedures, the titration curves obtained are in good agreement. 2 In this assay, the complement solution supplies an adequate amount of proteins to protect the target cells from harmful digestion. BSA (!%) may be used if less or no complement is present in the mixture. The cell number determined with the Coulter counter is linear at cell concentrations from 1 × 10 3 to 1 × 105 cells/ml. Application to Tissue Culture Cells
For this purpose the method is modified in two major points: Dispase is used instead of Pronase, and the Zap-Oglobin treatment is omitted. Thus, what is determined by this method is the number of cells with membranes that are resistant to dispase treatment. 5,6 Dispase (available 5 T. Matsumura, R. Konoshi, and Y. Nagai, In Vitro 18, 510 (1982). T. Matsumura and M. Nagata, Tissue Cult. 9, 348 (1983) (in Japanese).
[31]
CYTOTOXIC ASSAY WITH COULTER COUNTER
489
from Boehringer-Mannheim) is a neutral protease purified from Bacillus polymyxa. It is active and stable during incubation in a tissue culture medium containing fetal bovine serum. 7,8
Cell Preparations Cells, e.g., L-929 mouse fibroblasts in monolayer, and FM3A mouse mammary carcinoma cells in suspension, are cultivated in MEM supplemented with 5% (for FM3A cells) or 10% (for L cells) inactivated fetal bovine serum. A monodisperse suspension of live FM3A cells is obtained directly from the mother culture. L-cell monolayers are suspended either by mechanical dislodging, or by trypsinization, or by a dispase treatment using 1000 U (Anson's hemoglobin lysing unit) 7,8 dispase/ml in the growth medium. The cells are centrifuged and the pelleted cells are resuspended by pipetting. This cell suspension is called a live cell suspension. An aliquot of this suspension is frozen and thawed twice by alternatively putting it into a liquid nitrogen bath and a water bath. A dead cell suspension is thus obtained. The number of dye-excluding cells in both cell suspensions is determined by the use of erythrosin dye. 9 Aliquots of the live cell suspension and of the dead cell suspension are mixed, so that the mixed suspension contains 20 × 104 to 25 x 104 ceils/ml with the ratio of dye-excluding cells to total cells varying from less than 0.1% to more than 90%.
Dispase Treatment One milliliter of each of these mixed cell suspensions and 1 ml growth medium containing 2000 U/ml dispase are added to glass vials with 4.5 cm 2 bottom surface area (No. 986542, Wheaton Scientific). The vials are incubated for 30 min at 37° in a humidified atmosphere containing 5% CO2. After the incubation, 16 ml PBS is added to each vial, and the vials are kept in a ice-cold tray until electronic counting.
Electronic Counting Cells with a volume of more than 1.0 x 10 3 /zm 3 are enumerated as viable cells. In our counter, the lower threshold value, the upper threshold value, the aperture current value, and the amplification value were set at 21, 100, 4, and 2, respectively. 7 T. Matsumura, T. Yamanaka, S. Hashizume, Y. Irie, and K. Nitta, Jpn. J. Exp. Med. 45, 377 (1975). 8 T. Matsumura, K. Nitta, M. Yoshikawa, T. Takaoka, and H. Katsuta, Jpn. J. Exp. Med. 45, 383 (1975). 9 H. J. Phillips and R. V. Andrews, Exp. Cell Res. 16, 678 (1959).
490
ASSAYS FOR CYTOTOXIC ACTIVITY
[31]
Reliability and C o m m e n t s Optimal time of incubation with dispase is from 15 to 60 min. An electronic count value for a dead cell suspension, which contains less than 0.1% dye-excluding cells, is less than 5% (for L cells) or 2% (for FM3A cells) of that for the live cell suspension from which the dead cell suspension was prepared. The electronic count value and the percentage of dyeexcluding cells are proportional from 0.1% to more than 90% dye-excluding cells. For L cells, since the background value is relatively high, the following procedure is adopted. A vial containing the mixed cell suspension is incubated for a period sufficient for the live cells to attach to the bottom of the vials. The medium is then removed by suction and a l-ml aliquot of 1000 U/ml dispase solution is dispensed into each vial for a further 30 min incubation. The vials are placed for a few seconds onto a Vortex mixer to complete cell detachment. Then the cell suspensions are diluted with PBS and subjected to electronic counting as above. 5 Although the electronic counting method is simple and rapid, it also has a number of drawbacks. The number of viable cells determined by the present method and that determined by a dye-exclusion test, although they are proportional, differ considerably. The electronic count value may sometimes be only 70% of the value obtained by dye exclusion. This is because the electronic count value depends on the volume of the cells, which is affected by the culture conditions. Consequently, this method is less sensitive than a dye-exclusion test; the difference is at least a factor of 10. It is recommended that the electronic counting method be calibrated every time with a dye-exclusion test. The Coulter counter method is not generally applicable to any kind of tissue culture cells. It can be used for suspended cells and cells completely dissociable with dispase (e.g., L cells), but not for cells forming aggregates upon dispase treatment (e.g., HeLa cells). 8 The optimal concentration of dispase should be examined when other lines of cells are used, because treatment with high concentrations may sometimes lead to severe injury to the live cells.
CYTOTOXIC ASSAY WITH COULTER COUNTER
481
[31] Quantitative Cytotoxic Assay Using a Coulter Counter By Y O S H I T A K A
NAGAI, MACHIKO IWAMOTO, and
TOSHIHARU MATSUMURA
Cytotoxicity has been assayed either by determining the number of cells that become unable to exclude a dye, such as trypan blue, or by determining the amount of radioisotope released from preradiolabeled cells. The dye exclusion method is tedious and time consuming, but it is useful for a few samples. An unavoidable delay when many samples are handled causes an increase in the number of stained cells. Moreover, the presence of cell aggregates or erythrocytes causes interference. The radioisotope method is suitable to handle many samples, but it requires expensive instruments and reagents. Sometimes, spontaneous release of radioisotope complicates the analysis. It has been known that damaged cells are more susceptible to protease treatment than intact cells. To quantitate complement-mediated immune cytolysis of lymphosarcoma cells and mouse spleen cells, Stewart and Goldstein ~ combined the procedure of protease digestion with that of electronic counting. In this method, cells are first treated with a protease to completely digest damaged cells and subsequently with a detergent to lyse intact cells as well as erythrocytes. The number of residual nuclei from intact cells is determined electronically. We modified and extended the method of Stewart and Goldstein. This assay 2 is rapid and reproducible and gives results comparable with those given by a dye-exclusion method. As our objective was to develop a rapid and quantitative method for the determination of antibody-mediated cytotoxicity, we describe examples using thymocytes as target cells. This cytotoxicity assay, when appropriately modified, is applicable to the determination of viability of tissue culture cells. Therefore, examples using L-929 mouse fibroblasts and FM3A mouse mammary carcinoma cells are also described. Application to Antibody-Mediated Cytotoxicity
Reagents The medium used in all procedures is Eagle's MEM, adjusted to pH 7.2 with 7.5% NaHCO3. Pronase E (Kakenkagaku Company, Tokyo, C. C. Stewart and S. Goldstein, J. Lab. Clin. Med. 84, 425 (1974). 2 M. I w a m o t o and Y. Nagai, Jpn. J. Exp. Med. 51, 109 (1981).
METHODS IN ENZYMOLOGY,VOL. 132
Copyright © 1986by Academic Press, Inc. All rightsof reproductionin any form reserved.
482
ASSAYS FOR CYTOTOXIC ACTIVITY
[31]
Japan) 3 is dissolved in phosphate-buffered saline (PBS), 3a pH 7.2, at a concentration of 3 mg/ml and stored at - 2 0 ° in 2- to 3-ml aliquots. Pronase (purchased from Calbiochem) can be used as well as Pronase E. The frozen stock solution is thawed just before use and is filtered through a 0.45-/xm millipore filter for removal of interfering precipitates. Isoton II and Zap-Oglobin are purchased from Coulter Diagnostics (Hialeah, FL). Zap-Oglobin is a hemolysing agent for diagnostic use, which lyses erythrocytes and the membranes of nucleated cells to liberate the nuclei of leukocytes for counting. Trypan blue is dissolved in PBS at a concentration of 0.2%. Lyophilized guinea pig serum, dissolved in doubly distilled water and diluted 1 : 4 with medium, is used as complement after absorption with agar. 4 Although any medium appropriate for the culture of target cells may be used, addition of 10 mM HEPES and 1% BSA will prevent cell damage.
Target Cells Thymus of 5- to 8-week-old male C3H/He mouse is removed and teased apart with sharp forceps in medium. The cell suspension is pipetted repeatedly and then filtered through a stainless steel screen (200 mesh). For further details, see this series, Vol. 108 [6]. To obtain a 100% dead cell suspension, a portion of the thymocyte suspension is incubated at 56° for 30 min. The untreated portion is used as 100% viable cells. The two cell suspensions are diluted with medium to 5 × 106 ceils/ml, equal volumes of the two suspensions are mixed and used as 50% dead cells. Viability of these suspensions, measured with trypan blue dye exclusion, gave a value 2-7% dead cells for viable cells, 45-53% dead cells for 50% dead cells, and 100% dead cells for the 100% dead cell preparation.
Coulter Counter Setting In our experiments, a Model ZBI Coulter counter was used with a 100/~m orifice aperture tube. The common settings of the counter were as follows: manometer, 0.5 ml; matching switch, 20 K; gain trim, 0. The threshold settings for thymocyte nuclei were A (l/amplification), 1/2; I (1/aperture current), 0.354; TL (lower threshold), 15; To (upper threshold), 60 (nuclear threshold). The settings for intact thymocytes were A, 1; I, 1/2; TL, 15; Tu, 60 (cellular threshold). The K value of our Coulter counter was measured as 11.5, so that the volume difference per each threshold was 2.04/zm 3 for nuclear threshold and 5.75 p . m 3 for cellular threshold. 3 T h e n a m e Pronase E was c h a n g e d to Actinase E recently. 3a Abbreviations: PBS, phosphate-buffered saline; BSA, bovine s e r u m albumin. 4 A. C o h e n and M. Schlesinger, Transplantation 10, 130 (1970).
[31]
C Y T O T O X I C ASSAY W I T H C O U L T E R C O U N T E R
483
Analysis of Volume Frequency Distribution In order to obtain a precise counting of the target cells and an effective rejection of debris, the most important adjustment is the threshold setting. The volume distribution pattern of thymocyte nuclei is first examined to determine the threshold setting. Thymocyte suspensions diluted with Isoton II with or without Zap-Oglobin (three drops/10 ml of counting solution) are analyzed for volume frequency distribution. Figure 1A illustrates the volume distribution of a thymocyte suspension treated with Zap-Oglobin, and it is obtained from counting the number of particles in each threshold at a setting ofA = 1/2 and I = 0.354, which corresponds to 2.04 /xm3 per each threshold. In Fig. I A, two populations of particles are seen. The first distribution, showing a marked decrease from a threshold of 1 to 10, is due to debris. The second distribution, giving a peak at a threshold of about 30, is due to thymocyte nuclei. The mean volume of the nuclei was calculated to be 67 / z m 3. According to this distribution pattern, the particles which are counted between threshold 15 and threshold 60 are measured as thymocyte nuclei. On the other hand, the thymocyte suspension without Zap-Oglobin shows a similar distribution pattern at a setting A = 1 and I = 1/2, and the mean volume is 178/zm 3 (Fig. IB). The volume distribution pattern of erythrocytes (mean volume 77 tzm3) overlaps with that of thymocyte nuclei; erythrocytes, however, are completely solubilized by addition of Zap-Oglobin (Table I). Therefore, contaminating erythrocytes do not interfere in the cytotoxicity assay.
Recovery of Cell Counts after Treatment with Zap-Oglobin and Pronase E Addition of Zap-Oglobin after incubation with Pronase E results in the liberation of nuclei from the remaining viable cells. The effects of ZapOglobin and protease treatment on viable cells and dead cells are shown in Table I. Cells (5 x 105) of each suspension in 0.3 ml of medium containing 1% BSA are incubated with 0.3 ml of PBS or 0.3 ml of 3 mg/ml of Pronase E at 37°. After 30 min, 0.5 ml of the mixture is added to a counting vial containing 10 ml of Isoton II and is counted at cellular threshold (values under column headings 1 and 3, in Table I). Then, three drops (0.1 ml) of Zap-Oglobin are added to the vial and the sample is counted once more at nuclear threshold (values under column headings 2 and 4, in Table I). For the calculation of cell recovery after treatment with Zap-Oglobin and Pronase E, the complete recovery is set at the counts with ZapOglobin but without Pronase E treatment.
484
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ASSAYS FOR CYTOTOXIC ACTIVITY
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Threshold FIG. |. (A) Volume frequency distribution patterns of thymocyte nuclei. Sample treated with Isoton I1 and Zap-Oglobin. The mean volume of thymocyte nuclei (-~) is 67 /~m3. A = 0.5, I = 0.354, 2.04/~m 3 per each threshold. (B) Volume frequency distribution patterns of thymocytes (solid line), sample treated with lsoton 11 only, and thymocyte nuclei (broken line). The mean volume of thymocytes (,~) is 178/~m 3 and that of nuclei (,~) is 67/~m 3. A = !, ! = 0.5, 5.75/~m 3 per each threshold.
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ASSAYS FOR CYTOTOXIC ACTIVITY
TABLE II DOSE RESPONSE OF PRONASE E TREATMENT
Percentage decrease from Pronase E-free control Pronase E concentration (mg/ml)
Viable cells
50% Dead cells
100% Dead cells
0 1 2 3
0 0 1.0 0.4
0 39.5 43.2 48.5
0 89.5 88.5 96.1
T r y p a n blue a
5.0
50.0
100
a Percentageof dead cells estimated by the trypan blue dye exclusion method without Pronase E treatment.
In the case of a viable cell suspension, there is no difference in the counts for cells and nuclei, suggesting that the effect of protease is negligible (the percentage viability by dye exclusion is 97.2% and the percentage recovery with Pronase-Zap treatment is 94.7%). However, protease decreases the count of dead cells by about 80%. Addition of Zap-Oglobin causes the complete destruction of dead cells, yielding background level counts. When erythrocytes are present together with the target cells, they are completely solubilized with Zap-Oglobin, and so have no effect on the count of the target cells.
Effect of the Amount of Pronase E and of the Incubation Time The dose dependence of the effect of Pronase E on viable and dead cells is shown in Table II. Satisfactory results are obtained with Pronase E at 3 mg/ml. With this amount the observed lysis is 0.4% for viable cells, 48.5% for 50% dead cells, and 96.1% for 100% dead cells. Only partial digestion is observed at lower protease concentrations, and 5 mg/ml Pronase E has the same effect as 3 mg/ml Pronase E. The percentage of cells digested at 37° as a function of time does not change appreciably. After incubation at 37° for 30 min, only 5% of the viable cells are lost, but the percentage lysis of 100% dead cells is 98.5%, and that of 50% dead cells is 52.4%. Similar results are obtained after incubation for 60 min.
[31]
487
CYTOTOXIC ASSAY WITH COULTER COUNTER TABLE III REPRODUCIBILITY OF PRONASE E AND ZAP-OGLOBIN PROCEDURE Viable cells
Well number 1 2 3 4 5 Average SD c
Count displayed" 16,286 16,588 16,758 16,399 16,524
16,305 16,673 16,782 16,490 16,621
50% Dead cells
Number of cells/mlb 0 0 -4) 3.40 3.48 3.52 3.43 3.45 3.46 0.05
Count displayed 7,593 7,711 7,875 7,991 7,630
7,407 7,823 8,035 7,892 7,536
Number of cells/ml (10 4) 1.50 1.55 1.59 1.59 1.51 1.55 0.04
100% Dead cells
Count displayed 128 160 132 129 125
106 128 112 135 160
Number of cells/ml (10 -4) 0.02 0.03 0.02 0.03 0.03 0.03 0.01
a Number of particles in 0.5 ml of counting solution at a setting of A = 0.5, 1 = 0.354, TL = 15, Tu = 60.
b Sum of two displayed counts indicating cells/1 ml of counting solution. If the displayed count is over 10,000, the value is corrected by the available correction table of the Coulter counter. c Standard deviation.
Reproducibility of the Method To test the reproducibility of this method, five samples of each cell suspension are treated with 3 mg/ml of Pronase E at 37 ° for 30 min, then counted with Zap-Oglobin at the nuclear threshold. The results listed in Table III demonstrate the high reproducibility of this method. Furthermore, the counts of each cell suspension are stable for at least 3 hr, z which gives adequate time for measuring many samples at one time.
Assay Procedure of Antibody-Mediated Cytotoxicity Target cells (5 × 105 t h y m o c y t e s in 0. I ml of medium) are added to a plastic tube containing 0.1 ml of diluted antiserum and 0.1 ml of complement. Normal serum is present instead of antiserum in the controls. Each reaction mixture is incubated for 1 hr at 37 ° in a humidified atmosphere of 5% CO2-95% air. After incubation, 0.3 ml of Pronase E solution is added to each tube and the mixtures are further incubated for 30 min. At the end of the incubation, 0.5 ml of the final mixture from each tube is transferred to a counting vial containing 10 ml of Isoton II and three drops (0.1 ml) of Zap-Oglobin. After mixing, the nuclei in the solution are counted in a Coulter c o u n t e r at nuclear threshold. Counts displayed in the counter window are the n u m b e r of particles in 0.5 ml of counting solution. Counts/ 1 ml of the solution are calculated as the sum of the counts determined by
488
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ASSAYS FOR CYTOTOXIC ACTIVITY
5 x 106/ml thymocyte 0.1 ml diluted antiserum 0.1 ml complement x 5 0.1 ml /
i
(
~
,
I hr in 5% CO2 in a~r
0. 2% trypan blue 0.1ml
~'~ 3mg/mlPronaseE 0.3ml 370C, 30 min in 5% CO2 in air
Hemocytometer counting
0.5 ml sampling
~Isolon II 10 ml p Oglobm 0.1 ml
Dye Exclusion Method Coulter Counter counting Coulter Counter Method FIG. 2. Procedures for the dye-exclusion and Coulter counter methods.
counting the same sample twice. Results are expressed as follows: Percentage lysis (percentage decrease) = (I
counts in sample~ - counts in control/ 100
All tests are performed at least in triplicate. The procedures are summarized in Fig. 2, together with the dye-exclusion method. When a titration of anti-mouse brain-associated theta (0) serum with C3H/He mouse thymocyte is carried out by these two procedures, the titration curves obtained are in good agreement. 2 In this assay, the complement solution supplies an adequate amount of proteins to protect the target cells from harmful digestion. BSA (!%) may be used if less or no complement is present in the mixture. The cell number determined with the Coulter counter is linear at cell concentrations from 1 × 10 3 to 1 × 105 cells/ml. Application to Tissue Culture Cells
For this purpose the method is modified in two major points: Dispase is used instead of Pronase, and the Zap-Oglobin treatment is omitted. Thus, what is determined by this method is the number of cells with membranes that are resistant to dispase treatment. 5,6 Dispase (available 5 T. Matsumura, R. Konoshi, and Y. Nagai, In Vitro 18, 510 (1982). T. Matsumura and M. Nagata, Tissue Cult. 9, 348 (1983) (in Japanese).
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CYTOTOXIC ASSAY WITH COULTER COUNTER
489
from Boehringer-Mannheim) is a neutral protease purified from Bacillus polymyxa. It is active and stable during incubation in a tissue culture medium containing fetal bovine serum. 7,8
Cell Preparations Cells, e.g., L-929 mouse fibroblasts in monolayer, and FM3A mouse mammary carcinoma cells in suspension, are cultivated in MEM supplemented with 5% (for FM3A cells) or 10% (for L cells) inactivated fetal bovine serum. A monodisperse suspension of live FM3A cells is obtained directly from the mother culture. L-cell monolayers are suspended either by mechanical dislodging, or by trypsinization, or by a dispase treatment using 1000 U (Anson's hemoglobin lysing unit) 7,8 dispase/ml in the growth medium. The cells are centrifuged and the pelleted cells are resuspended by pipetting. This cell suspension is called a live cell suspension. An aliquot of this suspension is frozen and thawed twice by alternatively putting it into a liquid nitrogen bath and a water bath. A dead cell suspension is thus obtained. The number of dye-excluding cells in both cell suspensions is determined by the use of erythrosin dye. 9 Aliquots of the live cell suspension and of the dead cell suspension are mixed, so that the mixed suspension contains 20 × 104 to 25 x 104 ceils/ml with the ratio of dye-excluding cells to total cells varying from less than 0.1% to more than 90%.
Dispase Treatment One milliliter of each of these mixed cell suspensions and 1 ml growth medium containing 2000 U/ml dispase are added to glass vials with 4.5 cm 2 bottom surface area (No. 986542, Wheaton Scientific). The vials are incubated for 30 min at 37° in a humidified atmosphere containing 5% CO2. After the incubation, 16 ml PBS is added to each vial, and the vials are kept in a ice-cold tray until electronic counting.
Electronic Counting Cells with a volume of more than 1.0 x 10 3 /zm 3 are enumerated as viable cells. In our counter, the lower threshold value, the upper threshold value, the aperture current value, and the amplification value were set at 21, 100, 4, and 2, respectively. 7 T. Matsumura, T. Yamanaka, S. Hashizume, Y. Irie, and K. Nitta, Jpn. J. Exp. Med. 45, 377 (1975). 8 T. Matsumura, K. Nitta, M. Yoshikawa, T. Takaoka, and H. Katsuta, Jpn. J. Exp. Med. 45, 383 (1975). 9 H. J. Phillips and R. V. Andrews, Exp. Cell Res. 16, 678 (1959).
490
ASSAYS FOR CYTOTOXIC ACTIVITY
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Reliability and C o m m e n t s Optimal time of incubation with dispase is from 15 to 60 min. An electronic count value for a dead cell suspension, which contains less than 0.1% dye-excluding cells, is less than 5% (for L cells) or 2% (for FM3A cells) of that for the live cell suspension from which the dead cell suspension was prepared. The electronic count value and the percentage of dyeexcluding cells are proportional from 0.1% to more than 90% dye-excluding cells. For L cells, since the background value is relatively high, the following procedure is adopted. A vial containing the mixed cell suspension is incubated for a period sufficient for the live cells to attach to the bottom of the vials. The medium is then removed by suction and a l-ml aliquot of 1000 U/ml dispase solution is dispensed into each vial for a further 30 min incubation. The vials are placed for a few seconds onto a Vortex mixer to complete cell detachment. Then the cell suspensions are diluted with PBS and subjected to electronic counting as above. 5 Although the electronic counting method is simple and rapid, it also has a number of drawbacks. The number of viable cells determined by the present method and that determined by a dye-exclusion test, although they are proportional, differ considerably. The electronic count value may sometimes be only 70% of the value obtained by dye exclusion. This is because the electronic count value depends on the volume of the cells, which is affected by the culture conditions. Consequently, this method is less sensitive than a dye-exclusion test; the difference is at least a factor of 10. It is recommended that the electronic counting method be calibrated every time with a dye-exclusion test. The Coulter counter method is not generally applicable to any kind of tissue culture cells. It can be used for suspended cells and cells completely dissociable with dispase (e.g., L cells), but not for cells forming aggregates upon dispase treatment (e.g., HeLa cells). 8 The optimal concentration of dispase should be examined when other lines of cells are used, because treatment with high concentrations may sometimes lead to severe injury to the live cells.