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RNA synthesis in mouse spleen cells as a probe of antibody mediated cytotoxicity
Journal of Immunological Methods, 26 (1979) 75--86 © Elsevier/North-Holland Biomedical Press
75
RNA SYNTHESIS IN MOUSE SPLEEN CELLS AS A PROBE OF ANTIBODY MEDIATED CYTOTOXICITY
THOMAS J. STEPHENS and CAROL M. WARNER Department of Biochemistry and Biophysics, Iowa State University, Ames, IA 50011, U.S.A. (Received 18 May 1978, accepted 15 September 1978)
A new method of assaying antibody mediated cytotoxicity is described. The method is based on the observation that live spleen cells have a basal level of RNA synthetic activity, whereas dead spleen cells do not. Therefore, incorporation of [3H]uridine into high molecular weight material can be used as an index of the percentage of live cells in a cub ture. The advantages of measuring RNA synthesis rather than DNA synthesis ([3H]thymidine incorporation) for this purpose are that a short culturing time can be used and no mitogenic stimulation of the cultures is necessary. If spleen cells are killed with antispleen serum plus complement during a 1 h preincubation, they will not subsequently incorporate [3H]uridine into high molecular weight material. The number of cells, amount of radioactive label, and time of incubation have all been optimized for the RNA synthesis assay procedure. The RNA synthesis assay has been performed using spleen cells from 5 inbred strains of mice: A, C57BL/6, CBA, DBA/1 and SJL. The results obtained with this new assay procedure have been compared to results obtained with the trypan blue dye exclusion assay. The RNA synthesis assay gives identical results to the trypan blue dye exclusion assay, but has the advantages of being refractory to interference by a large number of dead cells, of being independent of operator subjectivity, and of being subject to automation.
INTRODUCTION Assays for antibody mediated cytotoxicity can be categorized into 5 c l a s s e s b a s e d u p o n t h e m e t h o d u s e d t o d i s t i n g u i s h live cells f r o m d e a d cells. I n t h e d y e e x c l u s i o n a s s a y ( G o r e r a n d O ' G o r m a n , 1 9 5 6 ; T e r a s a k i a n d M c C l e l l a n d , 1 9 6 4 ; W a l f o r d e t al., 1 9 6 4 ; M i t t a l e t al., 1 9 6 8 ; A m o s e t al., 1969) the cytotoxic effects of antibody and complement are measured by t h e f a i l u r e o f d e a d cells t o e x c l u d e v i t a l d y e s s u c h as t r y p a n b l u e o r e o s i n Y. Q u a n t i t a t i o n o f cells is d e t e r m i n e d b y m i c r o s c o p i c e x a m i n a t i o n o f t e s t s a m p l e s . A v a r i a t i o n o f t h e d y e e x c l u s i o n a s s a y is t h e use o f a f l u o r e s c e n t m a r k e r ( E d i d i n , 1 9 7 0 ; B r a w n e t al., 1 9 7 5 ) . I n t h i s a s s a y , e t h i d i u m b r o m i d e is e x c l u d e d f r o m v i a b l e c e l l s b u t r a p i d l y e n t e r s d a m a g e d c e l l s , in w h i c h it fluo r e s c e s . C y t o t o x i c i t y is e v a l u a t e d u s i n g a c o m m e r c i a l l y a v a i l a b l e f l u o r o m eter. A s e c o n d class o f c y t o t o x i c a s s a y is t h e d y e i n c l u s i o n a s s a y . I n t h e p r o c e -
76 dure of Filman et al. (1975), target cells, sensitive to antibody-dependent complement mediated cytotoxicity, undergo an intracellular delocalization of the supravital stain, neutral red. The results are determined by visual enumeration of cells which have accumulated the dye. A similar marker inclusion assay is the fluorochromasia c y t o t o x i c i t y assay (Rotman and Papermaster, 1966; Bodmer et al., 1967; Watanabe et al., 1971; MarcelliBarge et al., 1973). This assay is based upon the observation that free fluorescein, produced by intracellular esterase mediated hydrolysis of fluorescein diacetate, is retained by live cells but released from dead cells. Cell viability is determined by counting the number of fluorescent cells in a sample. In the third category, the colony inhibition assay (HellstrSm and SjSgren, 1965; HellstrSm and HellstrSm, 1971), target cells are grown in petri dishes or microtest plates prior to treatment with antibody and complement. Cells showing no sensitivity to the antiserum are enumerated by visual examination of cells or groups of cells forming colonies. The fourth category, represented by radioactive marker release assays, involves a pre-assay incubation step in which the radioisotope is concentrated intracellularly. SlCr-chromate (Sanderson, 1964; Wigzell, 1965; Holm and Perlmann, 1967; Brunner et al., 1968) or 86Rb-chloride (Chollet et al., 1974) are the most c o m m o n l y used labeling materials. Target cells are then incubated with antibody and complement, and a cytotoxic index determined by the activity released by the test sample divided by total activity released by control cells. In this paper we describe the development of a new c y t o t o x i c i t y assay for use on samples of resting mouse spleen cells which contain a large proportion of dead cells due to previous biochemical and immunological treatments. The assay was developed so that it would be refractory to interference by large numbers of dead cells, not require the prelabeling of target cells, be reasonably rapid, be independent of operator subjectivity, and be subject to automation. The dye exclusion and dye inclusion assays could not be used because of the interference by large numbers of dead cells. The colony formation assay could not be used because it requires a lengthy culturing period. The radioactive marker release assays were not suitable because of the requirement for the prelabeling of the target cells. The assay developed falls into a fifth category of cytotoxic assays which takes advantage of the fact that live cells will incorporate small molecular weight radioactive precursors into high molecular weight products, whereas dead cells will not. One such assay, which measures the incorporation of [3H]thymidine into DNA was first reported by Klein and Perlmann (1963), and later automated, using a MASH (multiple automated sample harvester), by Knudsen et al. (1974). This DNA synthesis assay procedure meets all the criteria set forth above, except the length of time required. In order to measure DNA synthesis the cells must be undergoing mitosis, and this requires mitogenic stimulation of the spleen cell culture, followed by a several day culture period. We decided to test whether the basal level of RNA synthetic
77 activity would be high enough in resting mouse spleen cells to use the incorporation of [~H]uridine into RNA as an index of the viability of a given cell population. The purpose of this paper is to describe this new assay procedure, and to compare it to the trypan blue dye exclusion cytotoxicity assay. MATERIALS AND METHODS
Mice All inbred strains of mice were obtained from the Jackson Laboratory, Bar Harbor, ME. The strains used in these studies were A (H-2a), C57BL/6 (H-2b), CBA (H-2k), DBA/1 (H-2 ~) and SJL (H-2S). All mice were females, from 6 to 8 months o f age.
Preparation of antisera Antisera were produced against whole spleen cells according to the m e t h o d of Batchelor (1973). Antiserum to CBA spleen cells was prepared by immunization of C57BL/6 mice. Reciprocal immunizations were used to produce antisera against C57BL/6 and A spleen cells, to eliminate crossreaction in cell mixing experiments. Thus, C57BL/6 mice were injected with A spleens, and A mice were injected with C57BL/6 spleens. Anti-H-2 sera for use on the DBA/1 mice (anti H-2 q) and on the SJL mice (anti/-/-2 ~) were obtained from Research Resources Branch, National Institutes of Health.
Cy totoxieity testing Antisera produced against spleen cells were evaluated using a trypan blue d y e exclusion assay (Warner et al., 1976) and by using the new R N A synthesis assay described below. In both assays, cells were isolated b y injecting 1.0 ml of RPMI 1640 with 25 mM Hepes buffer (ISI Biologicals, Cary, IL) into the spleen capsule and forcing the splenie contents through a 40 mesh stainless steel screen. Single cell suspensions were then prepared by passing clumps through a 1.0 ml plastic tuberculin syringe equipped with a 26-gauge needle. The cells were washed once, counted, and resuspended in RPMI 1640 with 25 mM Hepes buffer supplemented with 10% fetal calf serum, 1000 IU/ ml penicillin, 100 pg/ml streptomycin (Sigma Chemical Co., St. Louis, MO) and 1% of 200 mM glutamine (Gibco Co., New York). The final white blood cell concentration was adjusted to 6.0 X 106 cells/ml. Each assay tube received 20 pl of this suspension, to give 12 X 104 spleen white blood cells/ assay.
Trypan blue assay In each assay, 20 pl of the cell suspension were mixed with 20 pl of the appropriate serum dilution and 20 pl of a 1 : 4 dilution of fetal calf serum in 12 mm X 75 mm glass tubes. Then 20 pl of a 1 : 2 dilution of guinea pig serum (Miles Laboratory, Elkart, IN) were added as the source of complement. The guinea pig serum had been absorbed previously with agarose (Cohen and Schlesinger, 1970). The mixture was allowed to incubate at
78 37°C for 1 h in 7% CO2 in air, after which 20 pl of a 0.4% filtered trypan blue solution were added to each tube. After 3--5 rain, a minimum of 200 cells was assessed microscopically for viability. Each assay was performed in duplicate. R N A synthesis assay A n t i b o d y mediated cytotoxicity to spleen cells was evaluated by measuring RNA synthesis in target cells after treatment with antiserum and complement. In each assay, 20 pl of the 6.0 × 106 cells/ml suspension were combined with 20 pl of the appropriate serum dilution, 20 pl of a 1 : 4 dilution of fetal calf serum, and 20 pl of a 1 : 2 dilution of absorbed guinea pig serum. The assays were performed in 12 mm × 75 mm culture tubes fitted with caps (Falcon, Oxnard, CA). If the procedure were automated, as suggested later, the assays could be performed in 96-well microtiter plates. Tubes were incubated at 37°C in 7% CO2 in air. After this 1 h preincubation, 25 pl (500 pCi/ml) of a [3H]uridine (26.7 Ci/mmole, New England Nuclear, Boston, MA) solution were added. Simultaneously, 20 pl of supplemented culture medium were added to each tube. The supplemented culture medium served to maximize viability during labeling, and also to ensure a final volume of 150 pl for spotting on filter papers, as described below. The cells were then cultured for an additional 5 h, after which 25 pl of a solution containing yeast RNA (8 mg/ml) and 12% (w/v) deoxycholate in PBS (45 ° C) were added. The contents of the tubes were vigorously vortexed and spotted in 75 pl aliquots on DEAE-cellulose paper discs (Whatman DE81, 23 mm diameter, Clifton, NJ). Each tube was washed twice with 75 pl of a 0.01 M Tris, pH 7.4, 0.5% BSA solution and the washings spotted on two additional filter papers. Duplicate blanks consisted of tubes containing all ingredients, except spleen cells. The filters were then processed in a batch (the filters were numbered in pencil for identification) by the method of Litman (1968) as modified by Versteegh et al. (1975). The principle of the procedure is that DEAE-filter papers retain RNA by electrostatic attraction and matrix effects, while low molecular weight material (less than 10 nucleotides) is easily removed by extensive washing in phosphate buffer. The advantages of this procedure over conventional trichloroacetic acid precipitation on glass filter papers are that blank values can be reduced to less than 0.1% of the added radioactivity. Also, no unincorporated label is occluded by a large precipitate, and no high molecular weight material is removed by extensive washing. This means that recovery of small samples is quantitative and highly reproducible. Thus, the filters were washed extensively in 0.5 M sodium phosphate, pH 9.2, to remove unincorporated uridine. Following the final wash, the filters were quickly rinsed twice with distilled water, twice with 95% ethanol, once with ether and finally air dried. They were counted in a liquid scintillation counter, using 10 ml of a toluene fluor. The filter papers may be removed from the vials and the fluor reused several times.
79
Cytotoxic quantitation The percentage o f dead cells was calculated using the following formula: S
m
P- ~- CT BIB-~XIO0 where P = percentage o f dead cells in an assay; B = average cpm in blank tubes containing no spleen cells; S = average cpm in tubes containing an unknown sample of spleen cells and antiserum; C = average cpm observed in tubes with a theoretical 100% killing with antiserum. If the antiserum actually gives 100% killing o f cells, then C = B, and the d e n o m i n a t o r drops o u t of the equation; T = average cpm in control tubes containing spleen cells and normal mouse serum.
Experimental protocols To compare the sensitivity and accuracy o f the RNA syrithesis assay to the t r y p a n blue d y e exclusion assay, two types of experiments were designed. In the first type, the 5 antiserum samples were titered using a constant n u m b e r o f target cells. Supplemented culture medium was used as diluent for the antiserum samples. Increasing serum dilutions were chosen to reflect changes in c y t o t o x i c i t y and to permit the construction o f c y t o t o x i c killing curves for each o f the antiserum samples. C y t o t o x i c i t y was m o n i t o r e d using b o th the RNA synthesis assay and the t r y p a n blue dye exclusion assay. In the second t ype of experiment, the antiserum concent rat i on was kept constant, and the target cell concent r a t i on was varied. It should be n o t e d that the RNA synthesis assay measures the n u m b e r o f live cells in a population directly, regardless of the n u m b e r of dead cells present. T herefore various dilutions o f spleen white blood cells were made with supplemented RPMI 1640. Cells were treated with normal serum and c o m p l e m e n t for 1 h and the RNA synthetic activity measured, as previously described. The observed counts per minute (cpm) were used as an index o f the n u m b e r of live spleen white blood cells in a culture. In contrast, the t r y p a n blue dye exclusion assay measures the n u m b e r o f dead (or live) spleen white blood cells relative to the total n u m b e r o f cells present. Thus to get directly comparable results between the two assay procedures, the t rypan blue dye exclusion assay was set up as a series of mixing experiments. For example, A spleen white blood cells and C57BL/6 spleen white blood cells were mixed in varying p r o p o r tio n s so that the total cell n u m b e r in each assay would be constant. Thus, a culture tube might contain 9.6 X 1 0 4 A cells and 2.4 X 104 C57BL/6 cells, while a n o t h e r might contain 6.0 X 104 A cells and 6.0 X 104 C57BL/6 cells. The total cell n u m b e r in each assay was 12 X 104. Cells were treated with the appropriate antiserum and c o m p l e m e n t and the percentage o f dead cells determined. The cells selected for mixing experiments were
80 chosen to minimize nonspecific cytotoxicity of each antiserum sample with the other cell type. The percentage of dead cells killed by a specific antiserum would reflect the number of cells of one type in a mixture at the beginning of the experiment, and the results would be directly comparable to the viable cell number determined by the RNA synthesis assay. Various cell numbers of five different inbred strains of mice were analyzed using both the RNA synthesis assay and the trypan blue dye exclusion assay. RESULTS
(I) Analysis o f antisera Initially, each of the 5 antiserum samples was tested against whole spleen cell preparations using the trypan blue dye exclusion assay. All antisera were titered using a constant number of target cells (12 X 104 spleen white blood cells) in each assay tube. At a 1 : 16 antiserum dilution, it was found that 4 of the antisera killed t>95% of the correct target cells with less than 10% killing of control cells. The anti-H-2 q serum gave killing endpoint ranges of 70--90% in different experiments. None of the serum samples showed anticomplementary effects, when used with guinea pig serum as the source of complement. The 5 antiserum samples were used for all subsequent experiments.
(H) Conditions for R N A synthesis assay Fig. 1 shows the counts per minute (cpm) in 12 X 104 spleen cells (A strain) labeled for 5 h with varying amounts of [3H]uridine. Incorpora-
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81
tion into high molecular weight product was linear in the ranges 2.5--15 pCi [3H]uridine/assay. Cells labeled with amounts of [3H]uridine greater than 20 pCi showed decreased counts incorporated. A concentration of 12.5 pCi [ 3H]uridine/assay was chosen for all subsequent experiments. The optimal labeling period for spleen cells was 5 h, as is shown in Fig. 2. The incorporation of label into total RNA was linear for the 5 h period. Longer periods of labeling were found to be toxic to the cells. Thus, a 5 h labeling period was used for all subsequent experiments. Fig. 3 represents counts incorporated into spleen cells following different times of preincubation with either appropriate antiserum or normal serum and complement. Data are shown for only the A strain. The results with the other strains were similar. Cells were labeled for a 5 h period following the preincubation. In all experiments no detectable RNA synthesis was observed after a 1 h preincubation of cells with antiserum and complement. Samples treated with normal serum and complement showed good viability for a 2 h period, after which counts decreased. Thus, a I h preincubation period, and a 5 h labeling period with 12.5 pCi/assay [3H]uridine were the final conditions chosen for the RNA synthesis assay.
(III) Serum dilution experiments Fig. 4 demonstrates comparative changes in cytotoxicity in both the trypan blue dye exclusion and RNA synthesis assays as a result of increasing serum dilutions. The target cell number (12 × 104 spleen white blood cells/ assay) remained constant in these experiments. The titration curves are shown for only the A strain, since the results with the other strains were similar. The curves for all 5 strains were subjected to a computerized probit
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Fig. 3. O p t i m a l t i m e o f p r e i n c u b a t i o n , The cpm i n c o r p o r a t e d i n t o h i g h m o l e c u l a r w e i g h t p r o d u c t b y s p l e e n cells, i n c u b a t e d 5 h in t h e p r e s e n c e o f 1 2 . 5 p C i [ 3 H ] u r i d i n e p e r a s s a y , following different periods of preincubation with antiserum and complement. - cells t r e a t e d w i t h a 1 : 16 d i l u t i o n o f n o r m a l m o u s e s e r u m ; . . . . . . , cells t r e a t e d w i t h a 1 : 16 d i l u t i o n o f a n t i s e r u m . Fig. 4. A n a l y s i s o f s e r u m t i t e r . T h e p e r c e n t a g e o f d e a d cells as a f u n c t i o n o f r e c i p r o c a l d i l u t i o n s o f a n t i s e r u m . E a c h a s s a y w a s p e r f o r m e d as d e s c r i b e d in t h e t e x t u s i n g s p l e e n cells as t h e t a r g e t cells. D a t a are s h o w n f o r t h e R N A s y n t h e s i s a s s a y ( ) and the trypan blue dye exclusion assay ( ...... ).
analysis (Smith et al., 1977) to determine the 50% endpoint for both the RNA synthesis assay and the trypan blue dye exclusion assay. The results are shown in Table 1.
TABLE 1 ANTISERUM TITERS DETERMINED BY TRYPAN BLUE DYE EXCLUSION ASSAY a Antisera
Anti-A Anti-C57BL/6 Anti-CBA Anti-DBA/1 Anti-SJL
RNA
SYNTHESIS
ASSAY
AND
BY
Titer RNA synthesis assay
Trypan blue dye exclusion assay
71 537 100 209 158
71 468 138 166 182
a D a t a are t h e r e s u l t s o f a p r o b i t a n a l y s i s o f t h e t i t r a t i o n c u r v e s a c c o r d i n g to S m i t h et al. ( 1 9 7 7 ) . T h e v a l u e s are t h e c o m p u t e r c a l c u l a t e d r e c i p r o c a l s e r u m d i l u t i o n s giving a 50% cytotoxic endpoint.
83
(IV) Cell dilution experiments Fig. 5 shows the results of the RNA synthesis assay on various numbers of spleen cells from the A mouse strain. The other mouse strains gave similar results. The cpm incorporated reflect the number of live cells in each sample, so that an u n k n o w n sample may be analyzed by comparison with the standard curve. Similar samples were simultaneously evaluated using the trypan blue dye exclusion assay. Fig. 6 represents the analysis of these samples for the A strain. Again, the other strains gave similar results. As described in Materials and Methods, the samples used in these experiments contained mixtures of two cell types and were treated with antisera directed to one of the cell types. The percentage of dead cells was a reflection of the total number of cells of that type in the population. Table 2 shows a comparison of the data from the two assay procedures utilizing all the data collected from duplicate determinations on each of the 5 mouse strains. The estimated error in both the RNA synthesis and the trypan blue exclusion assays is less than 10%.
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1.2 2.4 3.64.8 6.0 7.2 8.4 9.6 10.812.0 A SWBC/ASSAY (x IU-4)
Fig. 5. Analysis o f R N A s y n t h e s i s assay. T h e w e i g h t p r o d u c t as a f u n c t i o n o f c o n c e n t r a t i o n regression line t h r o u g h t h e p o i n t s is s h o w n . T h e ity, P, t h a t t h e p o i n t s are n o t c o r r e l a t e d , have (1969).
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c p m i n c o r p o r a t e d i n t o high m o l e c u l a r o f spleen ceils. T h e least squares linear c o r r e l a t i o n coefficient, r, and p r o b a b i l b e e n c a l c u l a t e d a c c o r d i n g to B e v i n g t o n
Fig. 6. Analysis o f t r y p a n blue d y e exclusion assay. T h e p e r c e n t a g e o f dead cells as a f u n c t i o n o f c o n c e n t r a t i o n o f spleen cells. T h e least squares linear regression line t h r o u g h t h e p o i n t s is s h o w n . T h e c o r r e l a t i o n c o e f f i c i e n t , r, a n d p r o b a b i l i t y , P, t h a t t h e p o i n t s are n o t c o r r e l a t e d , have b e e n c a l c u l a t e d a c c o r d i n g to B e v i n g t o n ( 1 9 6 9 ) .
84 TABLE 2 C O M P A R I S O N OF R N A S Y N T H E S I S A S S A Y AND T R Y P A N B L U E DYE E X C L U S I O N ASSAY a Theoretical cell no. × 104
12.0 10.8 9.6 8.4 7.2 6.0 4.8 3.6 2.4 1.2
No. of determinations
10 10 10 10 10 10 10 10 10 10
E x p e r i m e n t a l cell no. × 104 R N A synthesis assay
T r y p a n blue dye exclusion assay
12.1±0.8 10.7±1.2 9.5±1.1 8.6±0.9 6.7±1.2 6.1 ± 1 . 0 4.7±1.0 3.7±0.9 2.2±0.5 1.2±0.4
11.7±0.6 10.7±0.6 9.6i0.9 9.0±0.7 7.9±0.4 6.3±1.0 5.0±0.9 3.1±0.9 2.3±0.6 2.1±2.7
a Data are c o m p i l e d f r o m d u p l i c a t e assays o f t h e A, C 5 7 B L / 6 , CBA, D B A / 1 , and SJL strains.
DISCUSSION
This study demonstrates that it is possible to evaluate antibody mediated c y t o t o x i c i t y by measuring RNA synthesis in target cells following incubation with antiserum and complement. Figs. 1 and 2 show that a 5 h labeling period employing 12.5 pC± of [3H]uridine is an optimal condition for measuring RNA synthesis in resting mouse spleen cells. This is in agreement with Cooper and Rubin (1965) who, in a series of experiments with resting lymphocytes, demonstrated t h a t incorporation of [~H]uridine increased over a period of 4--6 h and then reached a stable state beyond which incorporation decreased. Incorporation into high molecular weight product in their studies was linear up to a 5 h period with no change in the RNA content of the culture during this time. A 1 h preincubation period of cells with antiserum and complement was found adequate for stopping detectable RNA synthesis (Fig. 3). Control samples treated with normal mouse serum and complement for longer than a 2 h preincubation period had reduced viability. This is to be expected since guinea pig serum is known to contain some cytolysins (Sanderson, 1964). This could potentially be overcome by substituting rabbit serum as the source of complement. The analysis of all the data from the 5 mouse strains shows that the RNA synthesis assay gives statistically identical results to the trypan blue dye exclusion assay. In the RNA synthesis assay, there is a proportional relationship between the a m o u n t of label incorporated and the number of cells in
85 each assay. The assay can therefore be used to quantitate the number of live cells in an u n k n o w n cell population. Differences were observed in counts incorporated by identical numbers of cells from mice of different strains but these differences were found to reflect the uniqueness of each individual cell population, rather than to reflect consistent differences among mouse strains. Thus, the differences observed within one strain from day to day were as great as differences observed among strains. For this reason, control experiments must be included every time an assay is performed. Each of the currently available methods for evaluating antibody mediated c y t o t o x i c i t y has advantages and disadvantages. Among the c o m m o n l y used assays, the trypan blue assay has the advantages that it is inexpensive, fast, and requires few manipulations. The disadvantages are the possibility of operator subjectivity, a limit to the number of samples that can be analyzed by a single operator, and the impossibility of evaluating samples when a large percentage of dead cells are present. The S~Cr release assay has the advantage of being objective, but has the disadvantages of requiring the prelabeling and washing of cells. The DNA synthesis assay has the disadvantage of requiring mitogenic stimulation of the culture, and a long culturing period. Even though a large number of cytotoxicity assays have been described previously, none of the known c y t o t o x i c i t y assays met all the requirements for our work: no interference by large numbers of dead cells, no prelabeling of target cells, no long culturing periods, and no mitogen stimulation of the cultures. The presently described RNA synthesis assay does meet all the criteria set forth previously. First, there is no interference by dead cells, so that there is no need to separate dead cells nor red blood cells from the live spleen cell population, since neither of these have measurable levels of RNA synthesis. The RNA synthesis assay works as well on Ficoll-Hypaque isolated lymphocytes, and on t h y m o c y t e s , as on whole spleen cell suspensions (Stephens and Warner, unpublished). Second, the procedure does not require the extra manipulation of prelabeling the target cells. Third, the procedure can be done in hours instead of days as is required in the DNA synthesis assay. By increasing the specific activity of the [3H]uridine, the assay time could be cut down even farther. Fourth, the assay relies on the counting of radioactively labeled material, so it is independent of operator subjectivity. And finally, the procedure could be easily automated using microtiter plates and an automated cell harvester such as the MASH II (Microbiological Associates) or Titertek (Flow Laboratories, Scatron). The RNA synthesis cytotoxicity assay procedure should be applicable to any resting cells with a high basal level of RNA synthetic activity. ACKNOWLEDGEMENTS This work was supported by NIH Grant A I l 1 7 5 2 and NSF Grant PCM7715646.
86 W e t h a n k C a r l a M. T o l l e f s o n f o r t e c h n i c a l a s s i s t a n c e , t h e R e s e a r c h R e sources Branch, National Institutes of Health, for supplying the anti-H-2 sera, and Dr. David Cox, Iowa State University, for conducting the statistical analyses of the data. REFERENCES Amos, D.B., H. Bashir, W. Boyle, M. MacQueen and A. Tiilikainen, 1969, Transplantation 7, 220. Batchelor, J.R., 1973, in: Handbook of Experimental Immunology, ed. D.M. Weir (Blackwell Scientific Publications, Oxford) p. 32.1. Bevington, P.R., 1969, in: Data Reduction and Error Analysis for the Physical Sciences (McGraw-Hill, New York) p. 312. Bodmer, W., M. Tripp and J. Bodmer, 1967, in: Histocompatibility Testing, eds. E.S. Curtoni, P.L. Mattivz and R.M. Tosi (Williams and Wilkins, Baltimore, MD) p. 341. Brawn, R.J., C.R. Barker, A.D. Oesterle, R.J. Kelley and W.B. Dandliker, 1975, J. Immunol. Methods 9, 7. Brunner, K.T., J. Mauel, J.C. Cerottini and B. Chapuis, 1968, Immunology 14, 181. Chollet, Ph., J.M. Bidet, R. Plagne and B. Saurezie, 1974, J. Immunol. Methods 6, 23. Cohen, A. and M. Schlesinger, 1970, Transplantation 10, 130. Cooper, H. and A. Rubin, 1965, Blood 25, 1014. Edidin, M., 1970, J. Immunol. 104, 1303. Filman, D.J., R.J. Brawn and W.B. Dandliker, 1975, J. Immunol. Methods 6, 189. Gofer, P.A. and P. O'Gorman, 1956, Transplant. Bull. 3, 142. Hellstr6m, I. and K.E. HellstrSm, 1971, in: In Vitro Methods in Cell Mediated Immunity, eds. B.R. Bloom and P.R. Glade (Academic Press, New York) p. 409. Hellstr6m, I. and H.O. Sj6gren, 1965, Exp. Cell Res. 4 0 , 2 1 2 . Holm, G. and P. Perlmann, 1967, Immunology 12,525. Klein, G. and P. Perlmann, 1963, Nature 199,451. Knudsen, R.C., A.A. Ahmed and K.W. Sell, 1974, J. Immunol. Methods 5, 55. Litman, R.M., 1968, J. Biol. Chem. 243, 6222. Marcelli-Barge, A., A. Benajam, J.C. Poirier, L. Beraud and J. Dausset, 1973, IXe Congr. Natl. Transf. Sang., Nancy, 30 March--2 June. Mittal, K.K., M.R. Mickey, D.P. Singal and P.I. Terasaki, 1968, Transplantation 8, 913. Rotman, B. and B. Papermaster, ]966, Proc. Natl. Acad. Sci. U.S.A. 55, 134. Sanderson, A.R., 1964, Brit. J. Exp. Pathol. 45, 398. Smith, M.E., R. Laudico and B.W. Papermaster, 1977, J. Immunol. Methods 14, 243. Terasaki, P.I. and J.D. McClelland, 1964, Nature 2 0 4 , 9 2 8 . Versteegh, L.R., T.F. Hearn and C.M. Warner, ]975, Develop. Biol. 46,430. Walford, R.L., R. Gallagher and J.R. Sjaaraa, 1964, Science ] 4 4 , 8 6 8 . Warner, C.M., R.M. Graves, C.M. Tollefson, M.J.F. Schmerr, T.J. Stephens, C.F. Merryman and P.H. Maurer, 1976, Immunogenetics 3,337. Watanabe, T., Y. Yagi and D. Pressman, 1971, J. Immunol. 106, 1213. Wigzell, H., 1965, Transplantation 3,423.
75
RNA SYNTHESIS IN MOUSE SPLEEN CELLS AS A PROBE OF ANTIBODY MEDIATED CYTOTOXICITY
THOMAS J. STEPHENS and CAROL M. WARNER Department of Biochemistry and Biophysics, Iowa State University, Ames, IA 50011, U.S.A. (Received 18 May 1978, accepted 15 September 1978)
A new method of assaying antibody mediated cytotoxicity is described. The method is based on the observation that live spleen cells have a basal level of RNA synthetic activity, whereas dead spleen cells do not. Therefore, incorporation of [3H]uridine into high molecular weight material can be used as an index of the percentage of live cells in a cub ture. The advantages of measuring RNA synthesis rather than DNA synthesis ([3H]thymidine incorporation) for this purpose are that a short culturing time can be used and no mitogenic stimulation of the cultures is necessary. If spleen cells are killed with antispleen serum plus complement during a 1 h preincubation, they will not subsequently incorporate [3H]uridine into high molecular weight material. The number of cells, amount of radioactive label, and time of incubation have all been optimized for the RNA synthesis assay procedure. The RNA synthesis assay has been performed using spleen cells from 5 inbred strains of mice: A, C57BL/6, CBA, DBA/1 and SJL. The results obtained with this new assay procedure have been compared to results obtained with the trypan blue dye exclusion assay. The RNA synthesis assay gives identical results to the trypan blue dye exclusion assay, but has the advantages of being refractory to interference by a large number of dead cells, of being independent of operator subjectivity, and of being subject to automation.
INTRODUCTION Assays for antibody mediated cytotoxicity can be categorized into 5 c l a s s e s b a s e d u p o n t h e m e t h o d u s e d t o d i s t i n g u i s h live cells f r o m d e a d cells. I n t h e d y e e x c l u s i o n a s s a y ( G o r e r a n d O ' G o r m a n , 1 9 5 6 ; T e r a s a k i a n d M c C l e l l a n d , 1 9 6 4 ; W a l f o r d e t al., 1 9 6 4 ; M i t t a l e t al., 1 9 6 8 ; A m o s e t al., 1969) the cytotoxic effects of antibody and complement are measured by t h e f a i l u r e o f d e a d cells t o e x c l u d e v i t a l d y e s s u c h as t r y p a n b l u e o r e o s i n Y. Q u a n t i t a t i o n o f cells is d e t e r m i n e d b y m i c r o s c o p i c e x a m i n a t i o n o f t e s t s a m p l e s . A v a r i a t i o n o f t h e d y e e x c l u s i o n a s s a y is t h e use o f a f l u o r e s c e n t m a r k e r ( E d i d i n , 1 9 7 0 ; B r a w n e t al., 1 9 7 5 ) . I n t h i s a s s a y , e t h i d i u m b r o m i d e is e x c l u d e d f r o m v i a b l e c e l l s b u t r a p i d l y e n t e r s d a m a g e d c e l l s , in w h i c h it fluo r e s c e s . C y t o t o x i c i t y is e v a l u a t e d u s i n g a c o m m e r c i a l l y a v a i l a b l e f l u o r o m eter. A s e c o n d class o f c y t o t o x i c a s s a y is t h e d y e i n c l u s i o n a s s a y . I n t h e p r o c e -
76 dure of Filman et al. (1975), target cells, sensitive to antibody-dependent complement mediated cytotoxicity, undergo an intracellular delocalization of the supravital stain, neutral red. The results are determined by visual enumeration of cells which have accumulated the dye. A similar marker inclusion assay is the fluorochromasia c y t o t o x i c i t y assay (Rotman and Papermaster, 1966; Bodmer et al., 1967; Watanabe et al., 1971; MarcelliBarge et al., 1973). This assay is based upon the observation that free fluorescein, produced by intracellular esterase mediated hydrolysis of fluorescein diacetate, is retained by live cells but released from dead cells. Cell viability is determined by counting the number of fluorescent cells in a sample. In the third category, the colony inhibition assay (HellstrSm and SjSgren, 1965; HellstrSm and HellstrSm, 1971), target cells are grown in petri dishes or microtest plates prior to treatment with antibody and complement. Cells showing no sensitivity to the antiserum are enumerated by visual examination of cells or groups of cells forming colonies. The fourth category, represented by radioactive marker release assays, involves a pre-assay incubation step in which the radioisotope is concentrated intracellularly. SlCr-chromate (Sanderson, 1964; Wigzell, 1965; Holm and Perlmann, 1967; Brunner et al., 1968) or 86Rb-chloride (Chollet et al., 1974) are the most c o m m o n l y used labeling materials. Target cells are then incubated with antibody and complement, and a cytotoxic index determined by the activity released by the test sample divided by total activity released by control cells. In this paper we describe the development of a new c y t o t o x i c i t y assay for use on samples of resting mouse spleen cells which contain a large proportion of dead cells due to previous biochemical and immunological treatments. The assay was developed so that it would be refractory to interference by large numbers of dead cells, not require the prelabeling of target cells, be reasonably rapid, be independent of operator subjectivity, and be subject to automation. The dye exclusion and dye inclusion assays could not be used because of the interference by large numbers of dead cells. The colony formation assay could not be used because it requires a lengthy culturing period. The radioactive marker release assays were not suitable because of the requirement for the prelabeling of the target cells. The assay developed falls into a fifth category of cytotoxic assays which takes advantage of the fact that live cells will incorporate small molecular weight radioactive precursors into high molecular weight products, whereas dead cells will not. One such assay, which measures the incorporation of [3H]thymidine into DNA was first reported by Klein and Perlmann (1963), and later automated, using a MASH (multiple automated sample harvester), by Knudsen et al. (1974). This DNA synthesis assay procedure meets all the criteria set forth above, except the length of time required. In order to measure DNA synthesis the cells must be undergoing mitosis, and this requires mitogenic stimulation of the spleen cell culture, followed by a several day culture period. We decided to test whether the basal level of RNA synthetic
77 activity would be high enough in resting mouse spleen cells to use the incorporation of [~H]uridine into RNA as an index of the viability of a given cell population. The purpose of this paper is to describe this new assay procedure, and to compare it to the trypan blue dye exclusion cytotoxicity assay. MATERIALS AND METHODS
Mice All inbred strains of mice were obtained from the Jackson Laboratory, Bar Harbor, ME. The strains used in these studies were A (H-2a), C57BL/6 (H-2b), CBA (H-2k), DBA/1 (H-2 ~) and SJL (H-2S). All mice were females, from 6 to 8 months o f age.
Preparation of antisera Antisera were produced against whole spleen cells according to the m e t h o d of Batchelor (1973). Antiserum to CBA spleen cells was prepared by immunization of C57BL/6 mice. Reciprocal immunizations were used to produce antisera against C57BL/6 and A spleen cells, to eliminate crossreaction in cell mixing experiments. Thus, C57BL/6 mice were injected with A spleens, and A mice were injected with C57BL/6 spleens. Anti-H-2 sera for use on the DBA/1 mice (anti H-2 q) and on the SJL mice (anti/-/-2 ~) were obtained from Research Resources Branch, National Institutes of Health.
Cy totoxieity testing Antisera produced against spleen cells were evaluated using a trypan blue d y e exclusion assay (Warner et al., 1976) and by using the new R N A synthesis assay described below. In both assays, cells were isolated b y injecting 1.0 ml of RPMI 1640 with 25 mM Hepes buffer (ISI Biologicals, Cary, IL) into the spleen capsule and forcing the splenie contents through a 40 mesh stainless steel screen. Single cell suspensions were then prepared by passing clumps through a 1.0 ml plastic tuberculin syringe equipped with a 26-gauge needle. The cells were washed once, counted, and resuspended in RPMI 1640 with 25 mM Hepes buffer supplemented with 10% fetal calf serum, 1000 IU/ ml penicillin, 100 pg/ml streptomycin (Sigma Chemical Co., St. Louis, MO) and 1% of 200 mM glutamine (Gibco Co., New York). The final white blood cell concentration was adjusted to 6.0 X 106 cells/ml. Each assay tube received 20 pl of this suspension, to give 12 X 104 spleen white blood cells/ assay.
Trypan blue assay In each assay, 20 pl of the cell suspension were mixed with 20 pl of the appropriate serum dilution and 20 pl of a 1 : 4 dilution of fetal calf serum in 12 mm X 75 mm glass tubes. Then 20 pl of a 1 : 2 dilution of guinea pig serum (Miles Laboratory, Elkart, IN) were added as the source of complement. The guinea pig serum had been absorbed previously with agarose (Cohen and Schlesinger, 1970). The mixture was allowed to incubate at
78 37°C for 1 h in 7% CO2 in air, after which 20 pl of a 0.4% filtered trypan blue solution were added to each tube. After 3--5 rain, a minimum of 200 cells was assessed microscopically for viability. Each assay was performed in duplicate. R N A synthesis assay A n t i b o d y mediated cytotoxicity to spleen cells was evaluated by measuring RNA synthesis in target cells after treatment with antiserum and complement. In each assay, 20 pl of the 6.0 × 106 cells/ml suspension were combined with 20 pl of the appropriate serum dilution, 20 pl of a 1 : 4 dilution of fetal calf serum, and 20 pl of a 1 : 2 dilution of absorbed guinea pig serum. The assays were performed in 12 mm × 75 mm culture tubes fitted with caps (Falcon, Oxnard, CA). If the procedure were automated, as suggested later, the assays could be performed in 96-well microtiter plates. Tubes were incubated at 37°C in 7% CO2 in air. After this 1 h preincubation, 25 pl (500 pCi/ml) of a [3H]uridine (26.7 Ci/mmole, New England Nuclear, Boston, MA) solution were added. Simultaneously, 20 pl of supplemented culture medium were added to each tube. The supplemented culture medium served to maximize viability during labeling, and also to ensure a final volume of 150 pl for spotting on filter papers, as described below. The cells were then cultured for an additional 5 h, after which 25 pl of a solution containing yeast RNA (8 mg/ml) and 12% (w/v) deoxycholate in PBS (45 ° C) were added. The contents of the tubes were vigorously vortexed and spotted in 75 pl aliquots on DEAE-cellulose paper discs (Whatman DE81, 23 mm diameter, Clifton, NJ). Each tube was washed twice with 75 pl of a 0.01 M Tris, pH 7.4, 0.5% BSA solution and the washings spotted on two additional filter papers. Duplicate blanks consisted of tubes containing all ingredients, except spleen cells. The filters were then processed in a batch (the filters were numbered in pencil for identification) by the method of Litman (1968) as modified by Versteegh et al. (1975). The principle of the procedure is that DEAE-filter papers retain RNA by electrostatic attraction and matrix effects, while low molecular weight material (less than 10 nucleotides) is easily removed by extensive washing in phosphate buffer. The advantages of this procedure over conventional trichloroacetic acid precipitation on glass filter papers are that blank values can be reduced to less than 0.1% of the added radioactivity. Also, no unincorporated label is occluded by a large precipitate, and no high molecular weight material is removed by extensive washing. This means that recovery of small samples is quantitative and highly reproducible. Thus, the filters were washed extensively in 0.5 M sodium phosphate, pH 9.2, to remove unincorporated uridine. Following the final wash, the filters were quickly rinsed twice with distilled water, twice with 95% ethanol, once with ether and finally air dried. They were counted in a liquid scintillation counter, using 10 ml of a toluene fluor. The filter papers may be removed from the vials and the fluor reused several times.
79
Cytotoxic quantitation The percentage o f dead cells was calculated using the following formula: S
m
P- ~- CT BIB-~XIO0 where P = percentage o f dead cells in an assay; B = average cpm in blank tubes containing no spleen cells; S = average cpm in tubes containing an unknown sample of spleen cells and antiserum; C = average cpm observed in tubes with a theoretical 100% killing with antiserum. If the antiserum actually gives 100% killing o f cells, then C = B, and the d e n o m i n a t o r drops o u t of the equation; T = average cpm in control tubes containing spleen cells and normal mouse serum.
Experimental protocols To compare the sensitivity and accuracy o f the RNA syrithesis assay to the t r y p a n blue d y e exclusion assay, two types of experiments were designed. In the first type, the 5 antiserum samples were titered using a constant n u m b e r o f target cells. Supplemented culture medium was used as diluent for the antiserum samples. Increasing serum dilutions were chosen to reflect changes in c y t o t o x i c i t y and to permit the construction o f c y t o t o x i c killing curves for each o f the antiserum samples. C y t o t o x i c i t y was m o n i t o r e d using b o th the RNA synthesis assay and the t r y p a n blue dye exclusion assay. In the second t ype of experiment, the antiserum concent rat i on was kept constant, and the target cell concent r a t i on was varied. It should be n o t e d that the RNA synthesis assay measures the n u m b e r o f live cells in a population directly, regardless of the n u m b e r of dead cells present. T herefore various dilutions o f spleen white blood cells were made with supplemented RPMI 1640. Cells were treated with normal serum and c o m p l e m e n t for 1 h and the RNA synthetic activity measured, as previously described. The observed counts per minute (cpm) were used as an index o f the n u m b e r of live spleen white blood cells in a culture. In contrast, the t r y p a n blue dye exclusion assay measures the n u m b e r o f dead (or live) spleen white blood cells relative to the total n u m b e r o f cells present. Thus to get directly comparable results between the two assay procedures, the t rypan blue dye exclusion assay was set up as a series of mixing experiments. For example, A spleen white blood cells and C57BL/6 spleen white blood cells were mixed in varying p r o p o r tio n s so that the total cell n u m b e r in each assay would be constant. Thus, a culture tube might contain 9.6 X 1 0 4 A cells and 2.4 X 104 C57BL/6 cells, while a n o t h e r might contain 6.0 X 104 A cells and 6.0 X 104 C57BL/6 cells. The total cell n u m b e r in each assay was 12 X 104. Cells were treated with the appropriate antiserum and c o m p l e m e n t and the percentage o f dead cells determined. The cells selected for mixing experiments were
80 chosen to minimize nonspecific cytotoxicity of each antiserum sample with the other cell type. The percentage of dead cells killed by a specific antiserum would reflect the number of cells of one type in a mixture at the beginning of the experiment, and the results would be directly comparable to the viable cell number determined by the RNA synthesis assay. Various cell numbers of five different inbred strains of mice were analyzed using both the RNA synthesis assay and the trypan blue dye exclusion assay. RESULTS
(I) Analysis o f antisera Initially, each of the 5 antiserum samples was tested against whole spleen cell preparations using the trypan blue dye exclusion assay. All antisera were titered using a constant number of target cells (12 X 104 spleen white blood cells) in each assay tube. At a 1 : 16 antiserum dilution, it was found that 4 of the antisera killed t>95% of the correct target cells with less than 10% killing of control cells. The anti-H-2 q serum gave killing endpoint ranges of 70--90% in different experiments. None of the serum samples showed anticomplementary effects, when used with guinea pig serum as the source of complement. The 5 antiserum samples were used for all subsequent experiments.
(H) Conditions for R N A synthesis assay Fig. 1 shows the counts per minute (cpm) in 12 X 104 spleen cells (A strain) labeled for 5 h with varying amounts of [3H]uridine. Incorpora-
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81
tion into high molecular weight product was linear in the ranges 2.5--15 pCi [3H]uridine/assay. Cells labeled with amounts of [3H]uridine greater than 20 pCi showed decreased counts incorporated. A concentration of 12.5 pCi [ 3H]uridine/assay was chosen for all subsequent experiments. The optimal labeling period for spleen cells was 5 h, as is shown in Fig. 2. The incorporation of label into total RNA was linear for the 5 h period. Longer periods of labeling were found to be toxic to the cells. Thus, a 5 h labeling period was used for all subsequent experiments. Fig. 3 represents counts incorporated into spleen cells following different times of preincubation with either appropriate antiserum or normal serum and complement. Data are shown for only the A strain. The results with the other strains were similar. Cells were labeled for a 5 h period following the preincubation. In all experiments no detectable RNA synthesis was observed after a 1 h preincubation of cells with antiserum and complement. Samples treated with normal serum and complement showed good viability for a 2 h period, after which counts decreased. Thus, a I h preincubation period, and a 5 h labeling period with 12.5 pCi/assay [3H]uridine were the final conditions chosen for the RNA synthesis assay.
(III) Serum dilution experiments Fig. 4 demonstrates comparative changes in cytotoxicity in both the trypan blue dye exclusion and RNA synthesis assays as a result of increasing serum dilutions. The target cell number (12 × 104 spleen white blood cells/ assay) remained constant in these experiments. The titration curves are shown for only the A strain, since the results with the other strains were similar. The curves for all 5 strains were subjected to a computerized probit
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Fig. 3. O p t i m a l t i m e o f p r e i n c u b a t i o n , The cpm i n c o r p o r a t e d i n t o h i g h m o l e c u l a r w e i g h t p r o d u c t b y s p l e e n cells, i n c u b a t e d 5 h in t h e p r e s e n c e o f 1 2 . 5 p C i [ 3 H ] u r i d i n e p e r a s s a y , following different periods of preincubation with antiserum and complement. - cells t r e a t e d w i t h a 1 : 16 d i l u t i o n o f n o r m a l m o u s e s e r u m ; . . . . . . , cells t r e a t e d w i t h a 1 : 16 d i l u t i o n o f a n t i s e r u m . Fig. 4. A n a l y s i s o f s e r u m t i t e r . T h e p e r c e n t a g e o f d e a d cells as a f u n c t i o n o f r e c i p r o c a l d i l u t i o n s o f a n t i s e r u m . E a c h a s s a y w a s p e r f o r m e d as d e s c r i b e d in t h e t e x t u s i n g s p l e e n cells as t h e t a r g e t cells. D a t a are s h o w n f o r t h e R N A s y n t h e s i s a s s a y ( ) and the trypan blue dye exclusion assay ( ...... ).
analysis (Smith et al., 1977) to determine the 50% endpoint for both the RNA synthesis assay and the trypan blue dye exclusion assay. The results are shown in Table 1.
TABLE 1 ANTISERUM TITERS DETERMINED BY TRYPAN BLUE DYE EXCLUSION ASSAY a Antisera
Anti-A Anti-C57BL/6 Anti-CBA Anti-DBA/1 Anti-SJL
RNA
SYNTHESIS
ASSAY
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Titer RNA synthesis assay
Trypan blue dye exclusion assay
71 537 100 209 158
71 468 138 166 182
a D a t a are t h e r e s u l t s o f a p r o b i t a n a l y s i s o f t h e t i t r a t i o n c u r v e s a c c o r d i n g to S m i t h et al. ( 1 9 7 7 ) . T h e v a l u e s are t h e c o m p u t e r c a l c u l a t e d r e c i p r o c a l s e r u m d i l u t i o n s giving a 50% cytotoxic endpoint.
83
(IV) Cell dilution experiments Fig. 5 shows the results of the RNA synthesis assay on various numbers of spleen cells from the A mouse strain. The other mouse strains gave similar results. The cpm incorporated reflect the number of live cells in each sample, so that an u n k n o w n sample may be analyzed by comparison with the standard curve. Similar samples were simultaneously evaluated using the trypan blue dye exclusion assay. Fig. 6 represents the analysis of these samples for the A strain. Again, the other strains gave similar results. As described in Materials and Methods, the samples used in these experiments contained mixtures of two cell types and were treated with antisera directed to one of the cell types. The percentage of dead cells was a reflection of the total number of cells of that type in the population. Table 2 shows a comparison of the data from the two assay procedures utilizing all the data collected from duplicate determinations on each of the 5 mouse strains. The estimated error in both the RNA synthesis and the trypan blue exclusion assays is less than 10%.
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Fig. 5. Analysis o f R N A s y n t h e s i s assay. T h e w e i g h t p r o d u c t as a f u n c t i o n o f c o n c e n t r a t i o n regression line t h r o u g h t h e p o i n t s is s h o w n . T h e ity, P, t h a t t h e p o i n t s are n o t c o r r e l a t e d , have (1969).
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I
1.2 2.4 3,6 4.8 6.0 1.2 8.4-9.6 I0.812.0 A SWBC/ASSAY (x 10-4)
c p m i n c o r p o r a t e d i n t o high m o l e c u l a r o f spleen ceils. T h e least squares linear c o r r e l a t i o n coefficient, r, and p r o b a b i l b e e n c a l c u l a t e d a c c o r d i n g to B e v i n g t o n
Fig. 6. Analysis o f t r y p a n blue d y e exclusion assay. T h e p e r c e n t a g e o f dead cells as a f u n c t i o n o f c o n c e n t r a t i o n o f spleen cells. T h e least squares linear regression line t h r o u g h t h e p o i n t s is s h o w n . T h e c o r r e l a t i o n c o e f f i c i e n t , r, a n d p r o b a b i l i t y , P, t h a t t h e p o i n t s are n o t c o r r e l a t e d , have b e e n c a l c u l a t e d a c c o r d i n g to B e v i n g t o n ( 1 9 6 9 ) .
84 TABLE 2 C O M P A R I S O N OF R N A S Y N T H E S I S A S S A Y AND T R Y P A N B L U E DYE E X C L U S I O N ASSAY a Theoretical cell no. × 104
12.0 10.8 9.6 8.4 7.2 6.0 4.8 3.6 2.4 1.2
No. of determinations
10 10 10 10 10 10 10 10 10 10
E x p e r i m e n t a l cell no. × 104 R N A synthesis assay
T r y p a n blue dye exclusion assay
12.1±0.8 10.7±1.2 9.5±1.1 8.6±0.9 6.7±1.2 6.1 ± 1 . 0 4.7±1.0 3.7±0.9 2.2±0.5 1.2±0.4
11.7±0.6 10.7±0.6 9.6i0.9 9.0±0.7 7.9±0.4 6.3±1.0 5.0±0.9 3.1±0.9 2.3±0.6 2.1±2.7
a Data are c o m p i l e d f r o m d u p l i c a t e assays o f t h e A, C 5 7 B L / 6 , CBA, D B A / 1 , and SJL strains.
DISCUSSION
This study demonstrates that it is possible to evaluate antibody mediated c y t o t o x i c i t y by measuring RNA synthesis in target cells following incubation with antiserum and complement. Figs. 1 and 2 show that a 5 h labeling period employing 12.5 pC± of [3H]uridine is an optimal condition for measuring RNA synthesis in resting mouse spleen cells. This is in agreement with Cooper and Rubin (1965) who, in a series of experiments with resting lymphocytes, demonstrated t h a t incorporation of [~H]uridine increased over a period of 4--6 h and then reached a stable state beyond which incorporation decreased. Incorporation into high molecular weight product in their studies was linear up to a 5 h period with no change in the RNA content of the culture during this time. A 1 h preincubation period of cells with antiserum and complement was found adequate for stopping detectable RNA synthesis (Fig. 3). Control samples treated with normal mouse serum and complement for longer than a 2 h preincubation period had reduced viability. This is to be expected since guinea pig serum is known to contain some cytolysins (Sanderson, 1964). This could potentially be overcome by substituting rabbit serum as the source of complement. The analysis of all the data from the 5 mouse strains shows that the RNA synthesis assay gives statistically identical results to the trypan blue dye exclusion assay. In the RNA synthesis assay, there is a proportional relationship between the a m o u n t of label incorporated and the number of cells in
85 each assay. The assay can therefore be used to quantitate the number of live cells in an u n k n o w n cell population. Differences were observed in counts incorporated by identical numbers of cells from mice of different strains but these differences were found to reflect the uniqueness of each individual cell population, rather than to reflect consistent differences among mouse strains. Thus, the differences observed within one strain from day to day were as great as differences observed among strains. For this reason, control experiments must be included every time an assay is performed. Each of the currently available methods for evaluating antibody mediated c y t o t o x i c i t y has advantages and disadvantages. Among the c o m m o n l y used assays, the trypan blue assay has the advantages that it is inexpensive, fast, and requires few manipulations. The disadvantages are the possibility of operator subjectivity, a limit to the number of samples that can be analyzed by a single operator, and the impossibility of evaluating samples when a large percentage of dead cells are present. The S~Cr release assay has the advantage of being objective, but has the disadvantages of requiring the prelabeling and washing of cells. The DNA synthesis assay has the disadvantage of requiring mitogenic stimulation of the culture, and a long culturing period. Even though a large number of cytotoxicity assays have been described previously, none of the known c y t o t o x i c i t y assays met all the requirements for our work: no interference by large numbers of dead cells, no prelabeling of target cells, no long culturing periods, and no mitogen stimulation of the cultures. The presently described RNA synthesis assay does meet all the criteria set forth previously. First, there is no interference by dead cells, so that there is no need to separate dead cells nor red blood cells from the live spleen cell population, since neither of these have measurable levels of RNA synthesis. The RNA synthesis assay works as well on Ficoll-Hypaque isolated lymphocytes, and on t h y m o c y t e s , as on whole spleen cell suspensions (Stephens and Warner, unpublished). Second, the procedure does not require the extra manipulation of prelabeling the target cells. Third, the procedure can be done in hours instead of days as is required in the DNA synthesis assay. By increasing the specific activity of the [3H]uridine, the assay time could be cut down even farther. Fourth, the assay relies on the counting of radioactively labeled material, so it is independent of operator subjectivity. And finally, the procedure could be easily automated using microtiter plates and an automated cell harvester such as the MASH II (Microbiological Associates) or Titertek (Flow Laboratories, Scatron). The RNA synthesis cytotoxicity assay procedure should be applicable to any resting cells with a high basal level of RNA synthetic activity. ACKNOWLEDGEMENTS This work was supported by NIH Grant A I l 1 7 5 2 and NSF Grant PCM7715646.
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