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Microplate culture of mouse lymph node cells
Journal of Immunological Methods 5 (1974) 387-404. © North-HoUand Publishing Company
MICROPLATE CULTURE OF MOUSE LYMPH NODE CELLS I. Quantitation of Responses to Allogeneic Lymphocytes Endotoxin and Phytomitogens Philip E. THORPE and Stella C. KNIGHT Division of Surgical Sciences, ClinicalResearch Centre, Watford Road, Harrow, Middlesex, U.K.
Accepted 11 June 1974
Received 16 May 1974
A microplate culture system has been used to standardise mouse lymph node lymphocyte responses to concanavalin A (Con A), phytohaemagglutinin (PHA), pokeweed mitogen (PMW), endotoxin (LPS) and allogeneic lymphocytes and optimum culture and [3H]thymidine (3 H-Tdr) labelling conditions determined. The effects on lymphocyte transformation of microplate well-shape, foetal calf serum, cell concentration, mitogen dose, culture time, buffering system and initial pH of culture were examined. These factors showed different effects, both quantitative and qualitative, on the kinetics of the responses to the various stimulants resulting in dissimilar optimal culture conditions. This probably reflects either the different subpopulations of lymphocytes activated by these stimulants, or the different modes of action of the stimulants. Optimal 3H-Tdr labelling conditions were achieved with saturation concentrations of exogenous Tdr of at least 3.0 t~g/ml during culture in the most highly proliferating cultures. At saturation concentrations, any specific activity (SA) of 3H-Tdr could be used for quantitation since lymphocytes in different states of activation were affected by the radiobiological effects to the same extent. However, it was calculated from the parabolic relationship of 3H-Tdr incorporation and SA that at saturation concentrations the maximal SA that should be used to provide the highest uptake is 1.3 Ci/mM for a 24 hr pulse.
1. INTRODUCTION The 'in vitro' transformation of mouse lymphocytes in response to mitogens or allogeneic lymphocytes has become a common laboratory test in immunological research. PHA and Con A have been shown in rodents specifically to stimulate thymus-derived lymphocytes (T-cells). (Schrek and Batra, 1966; Rieke and Schwartz, 1967; Davies et al., 1968; Janossy and Greaves, 1972; Knight et al., 1973a). LPS specifically stimulates non-thymus processed lymphocytes (B-cells). (Andersson et al., 1972a; Gery et al., 1972) and PWM exhibits a mitogenic capacity for both T-cells and B-cells (Janossy and Greaves, 1972). The response to allogeneic lymphocytes in mixed lymphocyte culture (MLC) is another thymus-dependent 387
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P.E. THORPEand S.C. KNIGHT
response (Schwarz, 1967; Wilson et al., 1967; Knight et al., 1973a). T-cells are probably not homogenous and different subpopulations may be involved in the responses to various T-cell mitogens (Carr et al., 1973; Knight et al., 1973b; Stobo and Paul, 1973; Diamond et at., 1974). Some factors affecting microplate culture of human (Brody and Huntley, 1965; Parker and Lukes, 1971; Thurman et al., 1973) and rodent (Janossy and Greaves, 1972; Peck and Bach, 1973; Strong et al., 1973) lymphocytes have been described. This work confirms and extends observations on PHA- and Con A-stimulation of mouse lymphocytes in microculture, and provides new information on the use of LPS and allogeneic cells as stimulants. The importance of optimising the culture conditions and labelling technique for assessing lymphocyte transformation cannot be overstressed. A complete understanding of the variables involved is essential for interpretation of results. This study of many parameters in a single system demonstrates the considerable variations in the characteristics of subpopulations of lymphocytes responding to various stimulants.
2. MATERIALS AND METHODS 2.1. Mice
The mice used throughout this study were CBA/J (OLAC) female, aged 6 - 1 0 weeks. For the MCL's, C57BL/10 (OLAC) female mice provided the stimulating lymphocytes. 2.2. Cell suspensions
Mesenteric lymph nodes were removed aseptically from mice killed by cervical dislocation, and the cells gently teased through 100 gauge stainless steel mesh into 20 mM n-2-hydroxyethylpiperazine-n-2-ethane sulphonic acid (HEPES, Sigma,) buffered RPMI 1640 medium (Biocult) supplemented with glutamine 300 mM, penicillin 200 iu/ml, streptomycin 100 ~g/ml and 10% v/v heat-inactivated foetal calf serum (FCS, Biocult, batch 00262). Cell clumps and debris were allowed to settle for 10 min on ice before decanting the cell suspension. The suspension was centrifuged at 180g for 10min and the cells were resuspended in bicarbonate-buffered (25 mM) RPMI 1640 containing 10% FCS immediately before dispensing. Routinely, CBA cells were prepared at 3 × 10 6 viable lymphocytes per ml on the basis of Trypan Blue exclusion for stimulation with phytomitogens, at 8 × 10 6 per ml for LPS stimulation and at 10× 10 6 per ml for MLC. For undirectional MLC, C57BL/10 lymphocytes were inactivated either by treatment with mitomycin C (Nutritional Biochemicals, 25 tag/mt for 30 min at 37°C) and washing three times in HEPES-buffered medium, or by irradiation (1000 R, 6°Co source), before being prepared at 5 X 10 6 viable cells per ml.
Microplate culture of mouse lymph node cells
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2.3. Cell culture Microplate wells (excluding those at the edge of the plate where lower responses were found) received 0.2 ml volumes of cell suspensions (0.1 ml of each of two cell suspensions in the case of the MCL's) using a Hamilton repeating dispenser. The plates were routinely U-form microplates with loosely fitting lids (Cooke Engineering). The mitogens added (10/ll per culture) were phytohaemagglutinin (PHA-W, Burroughs Wellcome HA 15, 1:10 dilution of 5 ml reconstituted material), pokeweed mitogen (GIBCO 1/10 of 5 /~1 reconstituted material), concanavalin A (MilesYeda 1 ~g/well) and endotoxin (LPS-W, Difco). All cultures were set up in triplicate. Edge wells were filled with medium only and the plates incubated at 37°C in a humidified incubator gassed with 5% CO2 in air. Twenty four hours prior to harvesting 10/al of 3H.Tdr solution was added to each culture. For the majority of the PHA, Con A and PWM studies, 3 H-Tdr of SA 5 Ci/mM (Amersham, TRA 120) was diluted with cold Tdr (Koch Light) to give a solution of SA 150 mCi/mM and of total Tdr concentration 27.5 /ag/ml. For the MLC and LPS studies and a few of the PHA, Con A and PWM studies, 3H-Tdr solution was added of SA 370 mCi/mM and concentration 58/~g/ml. The SA of the Tdr used is indicated in each figure legend. Cultures were usually harvested after 72 hr. 2.4. Measurement o f pH pH measurements were made rapidly after equilibration at the required temperature in a water bath, using a Pye model 290 pH meter. 2.5. Harvesting procedure Microplates were centrifuged at 700g for 10 min. The supernatant was discarded, and each pellet was resuspended in 0.2 ml of saline. Similarly, two washes with 5% (w/v) trichloroacetic acid (TCA) and one wash with methanol were performed. After allowing the plates to dry, 0.2 ml of 1N NaOH were added to each well using an Eppendorf pipette, and the microplates were incubated for 30 min at 37°C in a humidified atmosphere to allow digestion of the TCA precipitable material. 0.1 ml of the digest from each well was then transferred to glass scintillation vials containing 1 ml methanol and 10 ml of scintillation fluid (2 parts xylene (BDH Chemicals) containing 0.6% (w/v) PPO (2.5 diphenyloxazole) and 0.012% (w/v)POPOP (p-bis (2-(5-phenyloxazolyl)-benzene), Nuclear Enterprises, to 1 part Triton X-100, BDH Chemicals). The vials were allowed to cold and dark adapt, and were counted on a Packard 'Tricarb' scintillation counter. Counts were quench corrected by means of an external standard-channels ratio technique to give disintegrations per minute (dpm).
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Fig. 1. The effect of welt shape upon the proliferative response of mouse lymphocytes to PHA as measured by the 3H-Tdr incorporation at different cell numbers per culture: A ~= unstimulated control for round-bottomed microplate and was closely similar to unstimulated control for flat-bottomed microplate. Pulsed with 3H-Tdr of SA 150 mCi/mM. Results are expressed as the arithmetic mean of triplicate determinations. Vertical bars on mean values in the figures represent one standard error o f mean, unless smaller than the points as plotted.
3. RESULTS
3.1. Effect of microplate well shape With round-bottomed wells, maximal stimulation was found using a lower cell concentration than with flat-bottomed wells. The round-bottomed wells allowed maximal stimulation with both PHA and Con A at 6 × 10 s cells/culture compared with 1.2 × 106 cells/culture using flat-bottomed wells (fig. 1). By virtue of the requirement for a lower cell number per culture, round b o t t o m e d plates were used in all subsequent experiments. Although there was no significant difference in the magnitude of stimulated or control values obtained with round or flat-bottomed wells at the optimal cell concentration in the experiment described by fig. 1, in general, flat-bottomed plates permitted higher optimal activity.
3.2. Relationship between cell concentration and optimal initial pH Aliquots o f HEPES-buffered (pH 6.5 at 25°C) cell suspensions at 3 or 6 × 106 cells/ml received 0.1 N NaOH to give a range o f pH values between 6.5 and 8.3 at 25°C. The stimulation in response to Con A was markedly pH dependent at b o t h cell concentrations (fig. 2). An initial pH o f 7.57 at 25°C (7.34 at 37°C) allows optimal stimulation with Con A at 6 X 10 s cells/culture. The higher cell concentra-
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Fig. 2. The effect of initial pH of culture medium (measured at 25°C) buffered with HEPES (20 raM) upon the 3H-Tdr incorporation of mouse lymphocytes in response to Con A. zx ~ = unstimulated control, closely similar for both cell concentrations. Pulsed with 3H-Tdr of SA 150 mCi/mM. tion o f 112 × 106 cells/culture required a more alkaline optimal pH value o f 8.0 at 25°C (7.77 at 37°C). The HEPES-buffered medium was subsequently prepared to a pH value o f 7.57 (at 25°C).
3.3. Comparison o f HEPES and bicarbonate buffers Cell suspensions were either prepared and cultured in HEPES-buffered medium or in bicarbonate-buffered medium, and these were compared with the routine technique o f preparing cell suspensions in the presence of HEPES buffer and culturing with bicarbonate buffer. With the cell concentrations used (0.2, 0.6 and 1.0 × 106 cells/culture) and with the stimulants used (PHA, Con A, PWM) bicarbonate buffer allowed at least 50% greater stimulation than with HEPES buffer. Manipulating the cells in HEPES-buffered medium before transfer to bicarbonatebuffered medium for culture (fig. 3) allowed a further increase in stimulation (p < 0.05). The latter technique was adopted routinely.
3.4. Effect o f variation in HEPES concentration The optimal HEPES concentration for Con A-stimulated cultures (initial pH 7.57 at 25°C) was 2 0 - 2 5 mM (fig. 4). Above 2 0 - 2 5 mM stimulation diminished gradually, probably due to toxicity of HEPES or the rise in tonicity. The effect was similar, but less marked, at the lower stimulation levels produced by PWM or suboptimal dosage of PHA (10 #l/culture of 1:20 dilution).
392
P.E. THORPE and S.C. KNIGHT
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Microplate culture of mouse lymph node cells
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3.5. Effect o f addition o f bicarbonate to HEPES-buffered medium A cell suspension was prepared in HEPES-buffered (16 mM) mediun supplemented with 5 mM bicarbonate (initial pH 7.57 at 25°C) and gassed with 1% CO2 which gave the correct partial pressure of CO2 necessary for the maintenance o f the o p t i m u m pH. The responses to Con A, PHA and PWM obtained with HEPESbuffered media supplemented with 5 mM bicarbonate were significantly increased (p < 0.05) compared to the responses obtained in HEPES-buffered media alone, but were still much inferior compared to responses obtained with bicarbonatebuffered media (table 1).
3. 6. The effect o f FCS concentration The FCS used throughout these studies (Biocult 00262) was selected from several batches for its ability to support stimulation without incurring high unstimulated control values. The optimal FCS concentration for support of the PHA response was 5 - 1 0 % . 10% FCS was used subsequently since this concentration produced a more consistent 3 H-Tdr incorporation in response to mitogens. Unstimulated control values were maximal between 15-20%.
3. 7. Effect o f cell concentration The optimal cell concentration for both PHA- and Con A-stimulated cultures was 6 × l 0 s cells per culture, whereas 1.5 to 2.0 × l 0 6 cells per culture was optimal for LPS-stimulated cultures (fig. 5). For optimal doses of these mitogerts, the effect o f cell number per culture on the response was marked. Suboptimal doses of PHA or Con A depressed the cell concentration curves in the.region of the maximal response, but otherwise showed similar kinetics. For optimal stimulation with PWM, the effect of cell concentration on the response was a plateau between 6 × l 0 s and 2.0 × 10 6 cells per culture (fig. 5b). In the absence of mitogen, cell Table I Effect of addition of sodium bicarbonate (5 raM) to HEPES (16 mM)-buffered medium on the 3H-Tdr incorporation of mouse lymphocytes in response to mitogens. Mitogen
None Con A PHA PWM
Buffer system (3H-Tdr incorporated, dpm -+S.E.) HEPES (20 mM)
Bicarbonate (25 mM)
H E P E S(16 mM) + Bicarbonate (5 mM)
620 41,160 38,950 4206
840 83.410 77,170 8170
820 50,490 45,720 4442
± 49 ± 1560 ± 1840 ± 315
Cultures pulsed with aH-Tdr of SA 370 mCi/mM.
± 92 ± 4212 ± 5176 ± 811
± 93 ± 2472 ± 2746 ± 202
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Fig. 5. The effect of cell concentration upon the 3H-Tdr incorporation of mouse lymphocytes in response to mitogens: (a) Optimal doses of Con A and PHA and a suboptimal dose of PHA (10 ~tl per culture of a 1:20 dilution), a a = unstimulated control; (b) Optimal doses of LPS and PWM; o. . . . . . o = actual dpm with LPS using 3H-Tdr of SA 370 mCi/mM; o - - - - - - o = calculated incorporation obtained with LPS using 3H-Tdr of SA 150 mCi/mM to facilitate comparison with the other mitogens; a A = unstimulated control. Pulsed with 3 H-Tdr of SA 150 mCi/mM (except o. . . . . . o). turnover was m ax i m u m with 1.6 to 2.0 × 106 cells/culture. In unidirectional MLC between CBA lymphocytes and irradiated C57 lymphocytes, the optimal cell concentrations were 1 X 106 cells/culture for the CBA population and 5 × 1 0 s cells/culture for the inactivated C57 population (fig. 6a). The magnitude of the unidirectional MLC response was markedly dependent upon the cell concentration o f the responding population, and less dependent upon the cell concentration o f the stimulating population. Syngeneic mixtures o f irradiated and non-irradiated CBA cells served as controls. The dose of irradiation (1000 R) was determined from a graph o f PHA response against radiation dose, and was the lowest dose that completely eliminated the PHA response. In each experiment, the unidirectional nature of the response was verified from the absence of response of the inactivated population to PHA. Mitomycin-C treatment as a means of inactivating the stimulating population allowed a greater stimulation and at a lower optimal stimulating cell concentration than did irradiation. (fig. 6b).
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Fig. 6. (a) The effect of the cell concentration of responding CBA lymphocytes upon the 3H-Tdr incorporation in response to unidirectional MLC with mitomycin-C-treated C57 BL/10 lymphocytes of cell concentrations 0.25, 0.5 and 0.75 X 106 cells per culture: • a = syngeneic unidirectional MLC with 0.5 X 106 mitomycin-C-treated CBA lymphocytes per culture; (b) The effect of the cell concentration of C57 BL/10 lymphocytes, inactivated by mitomycin-C or irradiation (1000 R) upon unidirectional MLC with 1.0 X 106 CBA lymphocytes per culture. Pulsed with 3H-Tdr of SA 370 mCi/mM.
3.8. Effect of mitogen dose The dose response curves for PHA and Con A were sharply peaked, whereas PWM and LPS produced broad dose response curves (fig. 7). The optimal doses of mitogens per culture were 1/ag of Con A, 5/ag of LPS, and 10/A of 1 : 10 dilution of PHA or PWM. The LPS was selected for maximal stimulation from several sources of endotoxin (table 2); all endotoxins tested produced optimal stimulation with 5 /lg/culture. Table 2 3H-Tdr incorporation of mouse lymphocytes in response to endotoxins from several sources. Endotoxin
3H-Tdr incorporation (dpm -+ S.E.)
None LPS-W;E. coli 0111 34 (Difco) LPS-W; S. typhimurium (Difco) LPS-W; S. enteriditis (Difco)
6,170 -+ 108 20,180 -+ 323 10,930 +- 570 19,020-+ 756 19,500 ± 54 16,700 -+ 517 17,670-+ 383
Escherichia coli* Salmonella typhimurium* Salmonella enteriditis*
Cultures were pulsed with 3H-Tdr of SA 370 mCi/mM. *These endotoxins were prepared by the method of Westphal et al. (1952), and were a gift from Dr. W.H. Adler.
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Fig. 7. The effect of mitogen dose upon the 3H-Tdr incorporation of mouse lymphocytes: (a) Stimulation by Con A and LPS: o. . . . . o = actual incorporation obtained with LPS using 3H-Tdr of SA 370 mCi/mM; o o = calculated incorporation obtained with LPS using 3H-Tdr of SA 150 mCi/mM to facilitate comparison with the other mitogens; (b) Stimulation with PHA and PWM. Mitogen dilution refers to the dilution of the 5 ml reconstituted material. Pulsed with 3H-Tdr of SA 150 mCi/mM (except o. . . . . o).
3.9. Effect of culture time The optimal culture time is dependent upon the initial cell concentration, higher cell concentrations favouring shorter culture times. This effect is shown for Con A in fig. 8a. The optimal culture period for Con A, PHA and PWM was 72 hr at the optimal cell concentration of 6 × 10 s cells per culture. For LPS, the optimal culture period was 24 hr earlier at 48 hr (fig. 8b). The magnitude of the undirectional MLC was less dependent upon the culture time than were the mitogen responses (fig. 9). As was apparent in the mitogen studies, a higher cell concentration favoured an earlier optimal culture period for MLC, although this relation was less marked. Maximal response in undirectional MLC was obtained between 4 8 - 7 2 hr of culture, with 1 to 1.5 X 106 responding cells per culture.
3.10. Preliminary comparison of mitogen responses of mesenteric lymph node, peripheral lymph nodes and spleen Cell suspensions were prepared from pooled mesenteric nodes, spleens, and from inguinal and axial nodes cultured under the optimal conditions as determined for mesenteric lymphocytes. Results are given in table 3. Splenic lymphocytes showed a greater responsiveness to LPS and PWM than either mesenteric or peripheral lymph node lymphocytes, whereas the responsiveness to PHA or Con A was not markedly different from lymph node lymphocytes. Unstimulated control values for
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Culture time (hours) Fig. 9. Effect of culture time on 3H-Tdr incorporation of mouse lymphocytes in response to unidirectional MLC. n D = response of 1.0 × 106 CBA lymphocytes per culture to 0.5 X 106 mitomycin-C-treated C57 BL/10 lymphocytes per culture, a ~ = syngeneic mixture of 1.0 X 106 CBA lymphocytes per culture and 0.5 X 106 mitomycin-C-treated lymphocytes per culture. Pulsed with 3 H-Tdr of SA 370 mCi/mM.
398
P.E. THORPE and S.C. KNIGHT
Table 3 Comparison of 3H-Tdr incorporation in response to mitogens in lymphocyte cultures derived from mesenteric lymph node, peripheral lymph node and spleen. Mitogen
None PHA Con A PWM LPS
3H-Tdr incorporation (mean dpm ± S.E.) Mesenteric lymph node
Peripheral lymph nodes
Spleen
1395 101,720 107,110 10,514 14,450
3287 102,210 139,170 16,950 11,630
8875 111,450 128,030 22,400 35,040
± 110 ± 3930 ± 3160 ± 870 ± 550
± 290 -+4920 ± 4070 ± 850 ± 930
± 545 ± 6460 ± 4090 ± 2590 ± 3290
Cultures were pulsed with 3H-Tdr of SA 370 mCi/mM.
splenic lymphocytes were considerably greater than for lymph node lymphocytes. The responses o f peripheral lymph node lymphocytes were fairly similar to those of mesenteric lymphocytes, the primary difference being in the 2-fold greater control value of the peripherally derived cells.
3.11. Effect o f concentration o f exogenous 3H- Tdr Ten microliters of 3H-Tdr solutions of varying concentrations from 5 to 160 pg/rnl, and o f constant low SA (150 mCi/mM) was added to identical cultures stimulated optimally with PHA, Con A or PWM. Cultures were harvested 24 hr later. Flooding conditions of exogenous 3H.Tdr were achieved for a 24 hr-pulse with 10/al/culture of Tdr solution o f concentration greater than 60/lg/ml (0.6/ag/ culture) for stimulation with Con A or PHA, and at greater than 30/ug/ml (0.3 ~ug/ culture) for stimulation with PWM (fig. 10). There was no indication that the Tdr concentration reached inhibitory levels at 1.6/ag/culture.
3.12. Effect o f specific activity o f 3H- Tdr Twenty microliters of aH.Tdr solutions of constant total Tdr concentration (27.5/ag/ml) and varying SA from 24 to 880 mCi/mM was added to identical cultures stimulated optimally with PHA, Con A or PWM. Cultures were harvested 24 hr later and results are shown in fig. 11. The relationship between SA and 3 H-Tdr incorporation is paraboloid. The mathematical equations of the parabolas obtained for PHA, Con A and PWM-stimulated cultures, and for unstimulated control cultures, were calculated from each individual value in fig. 11, and were seen to be mathematically similar. The SA that allows maximal a H-Tdr incorporation under saturation conditions with a 24 hr-pulse was calculated to be 1.3 Ci/mM. A SA o f 370 mCi/mM was chosen to enable easily measureable dpm to be obtained with weak stimulants.
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P.E. THORPE and S.C. KNIGHT
4. DISCUSSION The relationship between cell concentration in culture and proliferation in response to mitogens is influenced by such factors as availability of nutrients and mitogens, fall in pH and cell interactions (Ling, 1968). As these factors affect cultures with each stimulant, the differences in the relationship between cell concentration and proliferation for each stimulant probably reflect differences in the populations of cells being activated, and their mode of activation. Thus, the marked similarity in the kinetics of lymphocyte responses to Con A and PHA may indicate that they activate similar populations of cells by similar mechanisms. The proliferation in cultures stimulated with PWM is a plateau over a wide range of cell concentrations (compared with responses to Con A, LPS and optimal and suboptimal doses of PHA) possibly because PWM stimulates both T and B cells and effectively combines the Con A or PHA and the LPS response curves. This provides suggestive evidence that PWM causes independent stimulation of T and B cells. The importance of the shape of the culture vessel has been stressed previously for tube cultures (Moorhead et al., 1967; Ling, 1968) and for microplate cultures (Thurman et al., 1973). Round-bottomed wells were shown in this study to allow optimal stimulation at a lower cell concentration than flat-bottomed wells. It is probable that the choice of culture vessel affects the kinetics of the MLC response to a greater extent than mitogen responses due to the more conspicuous requirement for cell-cell contact (Chapman and Dutton, 1965; Moorhead et al., 1967). Hence, by comparison with mitogen studies, previous reports on the kinetics of the MLC response show greater variance (Adler et al., 1970; Mangi and Mardiney, 1970; Phillips et al., 1972; Peck and Bach, 1973). Consistently low PWM responses were found in this study compared with those obtained by other workers (Janossy and Greaves, 1971; Strong et al., 1973), and even under optimal conditions these only reached 10% of the PHA response. Lymphocytes from spleen and peripheral lymph nodes gave improved PWM responses compared to mesenteric lymphocytes, but these were still low at 21% and 16% of the PHA responses respectively. The mice used in this study were CBA female mice which have been reported to give considerably lower responses to PWM compared to CBA male mice (Strong et al., 1973). In humans, lymphocytes from males and females give similar PWM responses (unpublished observations). The log dose-response curves for Con A, PHA, PWM and LPS show approximately linear responses at dose levels below the optimum. For Con A and PHA, excess mitogen appears to be tolerogenic, since elution of the mitogen can enable recovery of the full response (Andersson et al., 1972b; Knight and Farrant, unpublished observations). Both LPS and PWM show a marked lack of either tolerogenicity or toxicity as shown by the very broad log dose-response curves. The optimal culture period for cells activated by a particular stimulant is probably determined by such factors as the characteristics of the target cell, the celt concentration and the magnitude of stimulation. Therefore, the earlier response to
Microplate culture o f mouse lymph node cells
401
LPS compared to Con A or PHA, may be a characteristic of the B-cell target, although PWM at a cell concentration of 3 × 106/ml does not show this earlier peak, or may merely be a manifestation of the high cell concentration required in LPS culture. Mitomycin-C treatment as a means of inactivating the stimulating population for unidirectional MLC allowed in a greater degree of activation, and at a lower optimal cell concentration that did irradiation. This was probably because cell death due to irradiation damage, although not initially obvious from trypan blue dye exclusion counts, occurred early during culture (Schrek, 1959). For lymphocyte culture, two buffers fulfil the necessary requirements of a pKa close to the optimum pH of the system, and low toxicity. These are sodium bicarbonate (pKa = 6.10 at 37°C, requiring equilibration with CO2 for maintenance of pH) and the sulphonic acid derivative HEPES (pKa = 7.31 at 37°C, Good et al., 1966). The buffering capacity of HEPES (20 raM) at the optimal culture pH of 7.34 is 1.1 X 10-2 g. equiv/pH unit compared to 0.27 X 10-2 g. equiv./pH unit for bicarbonate buffer (25 mM). These studies showed that bicarbonate buffer allowed at least 50% greater stimulation of mouse lymphocytes by mitogens than did HEPES buffer. Manipulation of the cells in HEPES-buffered medium before transfer to bicarbonate-buffered medium for culture overcame the gassing problem during handling, and allowed a further increase in stimulation. The stimulation obtained with HEPES buffer could not be improved by adjustment of HEPES concentration or initial pH. The small improvement in stimulation achieved upon addition of 5 mM bicarbonate approximated to the calculated increment allowed by the resulting mixed buffer system. Therefore, the inferiority of HEPES buffer compared to bicarbonate buffer was not due to an inferior buffering ability or to deficiency of essential bicarbonate ion. The possibility that HEPES was toxic to mouse lymphocytes was supported by the observation that even with low stimulation levels, such as with PWM and suboptimal PHA, when the demand for pH control is small HEPES was inferior to bicarbonate to the same extent as at high stimulation levels. Moreover, the small effect of the HEPES concentration upon cell proliferation (fig. 4) in view of the critical nature of the pH of culture (fig. 2) may indicate the interplay of an advantageous pH control effect and a disadvantageous effect of toxicity. Human lymphocytes responding to mitogen or MLC have been shown to allow equivalent stimulation with HEPES and bicarbonate buffers (Darzynkiewicz and Jacobson, 1971; Thurman et al., 1973)and Eagle (1971)showed that HEPES (20 raM) in combination with bicarbonate (24 raM) was not toxic to several mammalian cell types. Presumably mouse lymphocytes are unusually sensitive to HEPES toxicity. The optimal initial pH was dependent upon the initial cell concentration for cells stimulated with Con A, a higher cell concentration requiring a more alkaline pH. This is presumably because a greater cell number has a higher acid metabolite production, therefore an alkaline pH effectively neutralises the developing acidity. This is supported by preliminary data that indicates that addition of alkali to
402
P.E. THORPEand S.C. KNIGHT
cultures after 24 hr, as opposed to an initial change in pH, further increases stimulation in response to Con A in cultures of high cell concentration (1.2 × 106 per culture). The diminution of 3 H-Tdr uptake in Con A or PHA-stimulated cultures that is seen with cell concentration greater than 6 × l0 s cells per culture is, therefore, due, at least in part, to the fall in pH. The degree of activation of lymphocytes is assessed from the rate of DNA synthesis, as monitored by the incorporation of 3 H-Tdr. The concentration and SA of exogenous 3H-Tdr and the pulse time affect the correlation between rate of incorporation of label and rate of DNA synthesis (Cleaver, 1967). The pulse time of 24 hr was maintained throughout this study because the incorporation of label approaches linearity with pulse times of up to 24 hours at flooding concentrations of exogenous Tdr. (Schellekens and Eijsvoogel, 1968; Bain, 1970' Shons et al., 1972). This enables good incorporation of label to be achieved with 3 H-Tdr of low SA. The exogenous 3H_Tdr diffuses into the cell where it is phosphorylated to the mono-, di- or triphosphonucleotides (Bucher, 1963). The SA of these products is reduced by the dilutional effect of endogenous Tdr nucleotides. The incorporation of label into the DNA depends upon the resultant SA of the intracellular pool of Tdr triphosphate, and is maximal under conditions of excess exogenous Tdr. Under such saturation conditions the SA of the intracellular pool is constant during the 3 H-Tdr pulse, the % incorporation of label is small and volumetric errors during addition of 3H.Tdr solution are minimised. The closest correlation is therefore achieved between rate of incorporation of label and rate of DNA synthesis (Sample and Chretien, 1971 ; Quastler, 1963; Cleaver, 1967). In these studies, flooding conditions were achieved for a 24 hr-pulse with exogenous Tdr concentrations of 3.0/~g/ml in culture for optimal Con A and PHA stimulation. For lower levels of activation the % incorporation of exogenous Tdr is less, and flooding conditions are achieved with lower Tdr concentrations. At flooding Tdr concentrations, the SA of the intracellular pool ofTdr nucleotides is directly related to the SA of the exogenous 3 H-Tdr. However, the relationship between incorporation of label into DNA and the SA departs from linearity due to cellular inactivation from radiation damage and the percentage departure from linearity is proportional to the SA. Hence the rate of change of gradient in a graph of 3 H-Tdr incorporation versus SA is a constant, and double integration of this relationship gives the equation of a parabola. Mathematical treatment of each individual value for the dpm versus SA curves in fig. 11 enables the elaboration of the equations for each curve. The parabolic changes of dpm with SA for Con A, PHA and PWM and unstimulated control values are mathematically similar in spite of the markedly different degrees of activation. It is valid, therefore, for quantitation of the response to use any SA of 3 H-Tdr depending upon expense and required dpm, providing that flooding concentrations of Tdr are maintained throughout the pulse. The SA of 3 H-Tdr that gives maximal dpm with a 24 hr-pulse with saturation concentrations of Tdr can be calculated from fig. 11 to be 1.3 Ci/mM, and at
Microplate culture of mouse lymph node cells
403
greater SA the calculated dpm diminish. It is likely that the cellular inactivation due to the radiobiological effects is proportional to the pulse time, therefore for shorter pulses than 24 hr, the maximal SA can be increased proportionately.
ACKNOWLEDGEMENTS We gratefully acknowledge the help and discussion of Sir Peter Medawar, Dr. Noel Ling and Dr. Colin Sanderson. This work was supported by a grant from the Medical Research Council. REFERENCES Adler, W.H., T. Takiguchi, B. Marsh and R.T. Smith, 1970, J. Immunol. 105,984. Andersson, J., G. Mtiller and O. Sj6berg, 1972a, Cell. Immunol. 4, 381. Andersson, J., O. Sjoberg and G. MiSller, 1972b, Immunology 23,637. Bain, B., 1970, Clin. Exptl. Immunol. 6,255. Brody, J.A. and B. Huntley, 1965, Nature 208, 1232. Bucher, N.L.R., 1963, Intern. Rev. Cytol. 15,245. Carr, M.C., D.P. Stites and H.H. Fudenberg, 1973, Nature New Biol. 241,279. Chapman, N.D. and R.W. Dutton, 1965, J. Exptl. Med. 121,85. Cleaver, J.E., 1967, in: Thymidine metabolism and cell kinetics, eds. A. Neuberger and E.L. Tatum, (North Holland, Amsterdam.) Darzynkiewicz, Z. and B. Jacobson, 1971, Proc. Soc. Exptl. Biol. Med. 136,387. Davies, A.J.S., H. Festenstein, E. Leuchars, V.J. Wallisand M.J. Doenhoff, 1968, Lancet I, 183. Diamond, B., S.C. Knight and E.M. Lance, 1974, Cell. Immunol. II, 239. Eagle, H., 1971, Science 174,500. Gery, I., J. Kr'tigerand S.Z. Spiesel, 1972, J. Immunol. 108, 1088. Good, N.E., G.D. Winget, W. Winter, T.N. Connolly, S. Izawa and R.M.M. Singh, 1966, Biochemistry 5,467. Janossy, G., and M.F. Greaves, 1971, Clin. Exptl. Immunol. 9,483. Janossy, G. and M.F. Greaves, 1972, Clin. Exptl. Immunol. 10, 525. Knight, S.C., B. Newey and N.R. Ling, 1973a, Cytobios 7, 35. Knight, S.C., B. Newey and N.R. Ling, 1973b, Cell. Immunol. 9,273. Ling, N.R., 1968, in: Lymphocyte Stimulation (North Holland, Amsterdam). Mangi, R.J. and M.R. Mardiney, 1970, J. Immunol. 105, 90. Moorhead, J.F., J.J. Connally and W. McFarland, 1967, J. Immunol. 99,413. Parker, J.W. and R.J. Lukes, 1971, Amer. J. Clin. Pathol. 56,174. Peck, A.B. and F.H. Bach, 1973, J. Immunol. Methods 3,147. Phillips, S.M., C.B. Carpenter and J.P. Merrill, 1972, Cell. Immunol. 5,235. Quastler, H., 1963, in: Actions Chimiques et Biologiques des Radiations, ed. M. Haissinsky (Masson et Cie, Paris). Rieke, W.O. and M.R. Schwarz, 1967, Acta Haemat. 38,121. Sample, W.F. and P.B. Chretien, 1971, Clin. Exptl. Immunol. 9,419. Schellekens, P.Th.A. and V.P. Eijsvoogel, 1968, Clin. Exptl. Immunol. 3, 571. Schrek, R., 1959, Am. J. Clin. Pathol. 31,112. Schrek, R. and K.V. Batra, 1966, Lancet II, 444. Schwarz, M.R., 1967, Amer. J. Anat. 121,559.
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Shons, A.R., E.E. Etheredge and J.S. Najarian, 1972, Clin. Exptl. Immunol. 12,351. Stobo, J.D. and W.E. Paul, 1973, J. Immunol. 110, 362. Strong, D.M., A.A. Ahmed, G.B. Thurman and K.W. Sell, 1973, J. lmmunol. Methods 2,279. Thurman, G.B., D.M. Strong, A. Ahmed, S.S. Green, K.W. Sell, R.J. Hartzman and F.H. Bach, 1973, Clin. Exptl. Immunol. 15,289. Westphal, O., Liideritz and F. Bister, 1952, Z. Naturforsch Teil. B. 7b, 148. Wilson, D.B., W.K. Silvers and P.C. Nowell, 1967, J. Exptl. Med. 126,655.
MICROPLATE CULTURE OF MOUSE LYMPH NODE CELLS I. Quantitation of Responses to Allogeneic Lymphocytes Endotoxin and Phytomitogens Philip E. THORPE and Stella C. KNIGHT Division of Surgical Sciences, ClinicalResearch Centre, Watford Road, Harrow, Middlesex, U.K.
Accepted 11 June 1974
Received 16 May 1974
A microplate culture system has been used to standardise mouse lymph node lymphocyte responses to concanavalin A (Con A), phytohaemagglutinin (PHA), pokeweed mitogen (PMW), endotoxin (LPS) and allogeneic lymphocytes and optimum culture and [3H]thymidine (3 H-Tdr) labelling conditions determined. The effects on lymphocyte transformation of microplate well-shape, foetal calf serum, cell concentration, mitogen dose, culture time, buffering system and initial pH of culture were examined. These factors showed different effects, both quantitative and qualitative, on the kinetics of the responses to the various stimulants resulting in dissimilar optimal culture conditions. This probably reflects either the different subpopulations of lymphocytes activated by these stimulants, or the different modes of action of the stimulants. Optimal 3H-Tdr labelling conditions were achieved with saturation concentrations of exogenous Tdr of at least 3.0 t~g/ml during culture in the most highly proliferating cultures. At saturation concentrations, any specific activity (SA) of 3H-Tdr could be used for quantitation since lymphocytes in different states of activation were affected by the radiobiological effects to the same extent. However, it was calculated from the parabolic relationship of 3H-Tdr incorporation and SA that at saturation concentrations the maximal SA that should be used to provide the highest uptake is 1.3 Ci/mM for a 24 hr pulse.
1. INTRODUCTION The 'in vitro' transformation of mouse lymphocytes in response to mitogens or allogeneic lymphocytes has become a common laboratory test in immunological research. PHA and Con A have been shown in rodents specifically to stimulate thymus-derived lymphocytes (T-cells). (Schrek and Batra, 1966; Rieke and Schwartz, 1967; Davies et al., 1968; Janossy and Greaves, 1972; Knight et al., 1973a). LPS specifically stimulates non-thymus processed lymphocytes (B-cells). (Andersson et al., 1972a; Gery et al., 1972) and PWM exhibits a mitogenic capacity for both T-cells and B-cells (Janossy and Greaves, 1972). The response to allogeneic lymphocytes in mixed lymphocyte culture (MLC) is another thymus-dependent 387
388
P.E. THORPEand S.C. KNIGHT
response (Schwarz, 1967; Wilson et al., 1967; Knight et al., 1973a). T-cells are probably not homogenous and different subpopulations may be involved in the responses to various T-cell mitogens (Carr et al., 1973; Knight et al., 1973b; Stobo and Paul, 1973; Diamond et at., 1974). Some factors affecting microplate culture of human (Brody and Huntley, 1965; Parker and Lukes, 1971; Thurman et al., 1973) and rodent (Janossy and Greaves, 1972; Peck and Bach, 1973; Strong et al., 1973) lymphocytes have been described. This work confirms and extends observations on PHA- and Con A-stimulation of mouse lymphocytes in microculture, and provides new information on the use of LPS and allogeneic cells as stimulants. The importance of optimising the culture conditions and labelling technique for assessing lymphocyte transformation cannot be overstressed. A complete understanding of the variables involved is essential for interpretation of results. This study of many parameters in a single system demonstrates the considerable variations in the characteristics of subpopulations of lymphocytes responding to various stimulants.
2. MATERIALS AND METHODS 2.1. Mice
The mice used throughout this study were CBA/J (OLAC) female, aged 6 - 1 0 weeks. For the MCL's, C57BL/10 (OLAC) female mice provided the stimulating lymphocytes. 2.2. Cell suspensions
Mesenteric lymph nodes were removed aseptically from mice killed by cervical dislocation, and the cells gently teased through 100 gauge stainless steel mesh into 20 mM n-2-hydroxyethylpiperazine-n-2-ethane sulphonic acid (HEPES, Sigma,) buffered RPMI 1640 medium (Biocult) supplemented with glutamine 300 mM, penicillin 200 iu/ml, streptomycin 100 ~g/ml and 10% v/v heat-inactivated foetal calf serum (FCS, Biocult, batch 00262). Cell clumps and debris were allowed to settle for 10 min on ice before decanting the cell suspension. The suspension was centrifuged at 180g for 10min and the cells were resuspended in bicarbonate-buffered (25 mM) RPMI 1640 containing 10% FCS immediately before dispensing. Routinely, CBA cells were prepared at 3 × 10 6 viable lymphocytes per ml on the basis of Trypan Blue exclusion for stimulation with phytomitogens, at 8 × 10 6 per ml for LPS stimulation and at 10× 10 6 per ml for MLC. For undirectional MLC, C57BL/10 lymphocytes were inactivated either by treatment with mitomycin C (Nutritional Biochemicals, 25 tag/mt for 30 min at 37°C) and washing three times in HEPES-buffered medium, or by irradiation (1000 R, 6°Co source), before being prepared at 5 X 10 6 viable cells per ml.
Microplate culture of mouse lymph node cells
389
2.3. Cell culture Microplate wells (excluding those at the edge of the plate where lower responses were found) received 0.2 ml volumes of cell suspensions (0.1 ml of each of two cell suspensions in the case of the MCL's) using a Hamilton repeating dispenser. The plates were routinely U-form microplates with loosely fitting lids (Cooke Engineering). The mitogens added (10/ll per culture) were phytohaemagglutinin (PHA-W, Burroughs Wellcome HA 15, 1:10 dilution of 5 ml reconstituted material), pokeweed mitogen (GIBCO 1/10 of 5 /~1 reconstituted material), concanavalin A (MilesYeda 1 ~g/well) and endotoxin (LPS-W, Difco). All cultures were set up in triplicate. Edge wells were filled with medium only and the plates incubated at 37°C in a humidified incubator gassed with 5% CO2 in air. Twenty four hours prior to harvesting 10/al of 3H.Tdr solution was added to each culture. For the majority of the PHA, Con A and PWM studies, 3 H-Tdr of SA 5 Ci/mM (Amersham, TRA 120) was diluted with cold Tdr (Koch Light) to give a solution of SA 150 mCi/mM and of total Tdr concentration 27.5 /ag/ml. For the MLC and LPS studies and a few of the PHA, Con A and PWM studies, 3H-Tdr solution was added of SA 370 mCi/mM and concentration 58/~g/ml. The SA of the Tdr used is indicated in each figure legend. Cultures were usually harvested after 72 hr. 2.4. Measurement o f pH pH measurements were made rapidly after equilibration at the required temperature in a water bath, using a Pye model 290 pH meter. 2.5. Harvesting procedure Microplates were centrifuged at 700g for 10 min. The supernatant was discarded, and each pellet was resuspended in 0.2 ml of saline. Similarly, two washes with 5% (w/v) trichloroacetic acid (TCA) and one wash with methanol were performed. After allowing the plates to dry, 0.2 ml of 1N NaOH were added to each well using an Eppendorf pipette, and the microplates were incubated for 30 min at 37°C in a humidified atmosphere to allow digestion of the TCA precipitable material. 0.1 ml of the digest from each well was then transferred to glass scintillation vials containing 1 ml methanol and 10 ml of scintillation fluid (2 parts xylene (BDH Chemicals) containing 0.6% (w/v) PPO (2.5 diphenyloxazole) and 0.012% (w/v)POPOP (p-bis (2-(5-phenyloxazolyl)-benzene), Nuclear Enterprises, to 1 part Triton X-100, BDH Chemicals). The vials were allowed to cold and dark adapt, and were counted on a Packard 'Tricarb' scintillation counter. Counts were quench corrected by means of an external standard-channels ratio technique to give disintegrations per minute (dpm).
390
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Fig. 1. The effect of welt shape upon the proliferative response of mouse lymphocytes to PHA as measured by the 3H-Tdr incorporation at different cell numbers per culture: A ~= unstimulated control for round-bottomed microplate and was closely similar to unstimulated control for flat-bottomed microplate. Pulsed with 3H-Tdr of SA 150 mCi/mM. Results are expressed as the arithmetic mean of triplicate determinations. Vertical bars on mean values in the figures represent one standard error o f mean, unless smaller than the points as plotted.
3. RESULTS
3.1. Effect of microplate well shape With round-bottomed wells, maximal stimulation was found using a lower cell concentration than with flat-bottomed wells. The round-bottomed wells allowed maximal stimulation with both PHA and Con A at 6 × 10 s cells/culture compared with 1.2 × 106 cells/culture using flat-bottomed wells (fig. 1). By virtue of the requirement for a lower cell number per culture, round b o t t o m e d plates were used in all subsequent experiments. Although there was no significant difference in the magnitude of stimulated or control values obtained with round or flat-bottomed wells at the optimal cell concentration in the experiment described by fig. 1, in general, flat-bottomed plates permitted higher optimal activity.
3.2. Relationship between cell concentration and optimal initial pH Aliquots o f HEPES-buffered (pH 6.5 at 25°C) cell suspensions at 3 or 6 × 106 cells/ml received 0.1 N NaOH to give a range o f pH values between 6.5 and 8.3 at 25°C. The stimulation in response to Con A was markedly pH dependent at b o t h cell concentrations (fig. 2). An initial pH o f 7.57 at 25°C (7.34 at 37°C) allows optimal stimulation with Con A at 6 X 10 s cells/culture. The higher cell concentra-
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Fig. 2. The effect of initial pH of culture medium (measured at 25°C) buffered with HEPES (20 raM) upon the 3H-Tdr incorporation of mouse lymphocytes in response to Con A. zx ~ = unstimulated control, closely similar for both cell concentrations. Pulsed with 3H-Tdr of SA 150 mCi/mM. tion o f 112 × 106 cells/culture required a more alkaline optimal pH value o f 8.0 at 25°C (7.77 at 37°C). The HEPES-buffered medium was subsequently prepared to a pH value o f 7.57 (at 25°C).
3.3. Comparison o f HEPES and bicarbonate buffers Cell suspensions were either prepared and cultured in HEPES-buffered medium or in bicarbonate-buffered medium, and these were compared with the routine technique o f preparing cell suspensions in the presence of HEPES buffer and culturing with bicarbonate buffer. With the cell concentrations used (0.2, 0.6 and 1.0 × 106 cells/culture) and with the stimulants used (PHA, Con A, PWM) bicarbonate buffer allowed at least 50% greater stimulation than with HEPES buffer. Manipulating the cells in HEPES-buffered medium before transfer to bicarbonatebuffered medium for culture (fig. 3) allowed a further increase in stimulation (p < 0.05). The latter technique was adopted routinely.
3.4. Effect o f variation in HEPES concentration The optimal HEPES concentration for Con A-stimulated cultures (initial pH 7.57 at 25°C) was 2 0 - 2 5 mM (fig. 4). Above 2 0 - 2 5 mM stimulation diminished gradually, probably due to toxicity of HEPES or the rise in tonicity. The effect was similar, but less marked, at the lower stimulation levels produced by PWM or suboptimal dosage of PHA (10 #l/culture of 1:20 dilution).
392
P.E. THORPE and S.C. KNIGHT
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3.5. Effect o f addition o f bicarbonate to HEPES-buffered medium A cell suspension was prepared in HEPES-buffered (16 mM) mediun supplemented with 5 mM bicarbonate (initial pH 7.57 at 25°C) and gassed with 1% CO2 which gave the correct partial pressure of CO2 necessary for the maintenance o f the o p t i m u m pH. The responses to Con A, PHA and PWM obtained with HEPESbuffered media supplemented with 5 mM bicarbonate were significantly increased (p < 0.05) compared to the responses obtained in HEPES-buffered media alone, but were still much inferior compared to responses obtained with bicarbonatebuffered media (table 1).
3. 6. The effect o f FCS concentration The FCS used throughout these studies (Biocult 00262) was selected from several batches for its ability to support stimulation without incurring high unstimulated control values. The optimal FCS concentration for support of the PHA response was 5 - 1 0 % . 10% FCS was used subsequently since this concentration produced a more consistent 3 H-Tdr incorporation in response to mitogens. Unstimulated control values were maximal between 15-20%.
3. 7. Effect o f cell concentration The optimal cell concentration for both PHA- and Con A-stimulated cultures was 6 × l 0 s cells per culture, whereas 1.5 to 2.0 × l 0 6 cells per culture was optimal for LPS-stimulated cultures (fig. 5). For optimal doses of these mitogerts, the effect o f cell number per culture on the response was marked. Suboptimal doses of PHA or Con A depressed the cell concentration curves in the.region of the maximal response, but otherwise showed similar kinetics. For optimal stimulation with PWM, the effect of cell concentration on the response was a plateau between 6 × l 0 s and 2.0 × 10 6 cells per culture (fig. 5b). In the absence of mitogen, cell Table I Effect of addition of sodium bicarbonate (5 raM) to HEPES (16 mM)-buffered medium on the 3H-Tdr incorporation of mouse lymphocytes in response to mitogens. Mitogen
None Con A PHA PWM
Buffer system (3H-Tdr incorporated, dpm -+S.E.) HEPES (20 mM)
Bicarbonate (25 mM)
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840 83.410 77,170 8170
820 50,490 45,720 4442
± 49 ± 1560 ± 1840 ± 315
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± 92 ± 4212 ± 5176 ± 811
± 93 ± 2472 ± 2746 ± 202
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Fig. 5. The effect of cell concentration upon the 3H-Tdr incorporation of mouse lymphocytes in response to mitogens: (a) Optimal doses of Con A and PHA and a suboptimal dose of PHA (10 ~tl per culture of a 1:20 dilution), a a = unstimulated control; (b) Optimal doses of LPS and PWM; o. . . . . . o = actual dpm with LPS using 3H-Tdr of SA 370 mCi/mM; o - - - - - - o = calculated incorporation obtained with LPS using 3H-Tdr of SA 150 mCi/mM to facilitate comparison with the other mitogens; a A = unstimulated control. Pulsed with 3 H-Tdr of SA 150 mCi/mM (except o. . . . . . o). turnover was m ax i m u m with 1.6 to 2.0 × 106 cells/culture. In unidirectional MLC between CBA lymphocytes and irradiated C57 lymphocytes, the optimal cell concentrations were 1 X 106 cells/culture for the CBA population and 5 × 1 0 s cells/culture for the inactivated C57 population (fig. 6a). The magnitude of the unidirectional MLC response was markedly dependent upon the cell concentration o f the responding population, and less dependent upon the cell concentration o f the stimulating population. Syngeneic mixtures o f irradiated and non-irradiated CBA cells served as controls. The dose of irradiation (1000 R) was determined from a graph o f PHA response against radiation dose, and was the lowest dose that completely eliminated the PHA response. In each experiment, the unidirectional nature of the response was verified from the absence of response of the inactivated population to PHA. Mitomycin-C treatment as a means of inactivating the stimulating population allowed a greater stimulation and at a lower optimal stimulating cell concentration than did irradiation. (fig. 6b).
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0.25 0 . 5 0 0 . 7 5 1.00 Number of C57 BL/10 cells per culture x 10-6
Fig. 6. (a) The effect of the cell concentration of responding CBA lymphocytes upon the 3H-Tdr incorporation in response to unidirectional MLC with mitomycin-C-treated C57 BL/10 lymphocytes of cell concentrations 0.25, 0.5 and 0.75 X 106 cells per culture: • a = syngeneic unidirectional MLC with 0.5 X 106 mitomycin-C-treated CBA lymphocytes per culture; (b) The effect of the cell concentration of C57 BL/10 lymphocytes, inactivated by mitomycin-C or irradiation (1000 R) upon unidirectional MLC with 1.0 X 106 CBA lymphocytes per culture. Pulsed with 3H-Tdr of SA 370 mCi/mM.
3.8. Effect of mitogen dose The dose response curves for PHA and Con A were sharply peaked, whereas PWM and LPS produced broad dose response curves (fig. 7). The optimal doses of mitogens per culture were 1/ag of Con A, 5/ag of LPS, and 10/A of 1 : 10 dilution of PHA or PWM. The LPS was selected for maximal stimulation from several sources of endotoxin (table 2); all endotoxins tested produced optimal stimulation with 5 /lg/culture. Table 2 3H-Tdr incorporation of mouse lymphocytes in response to endotoxins from several sources. Endotoxin
3H-Tdr incorporation (dpm -+ S.E.)
None LPS-W;E. coli 0111 34 (Difco) LPS-W; S. typhimurium (Difco) LPS-W; S. enteriditis (Difco)
6,170 -+ 108 20,180 -+ 323 10,930 +- 570 19,020-+ 756 19,500 ± 54 16,700 -+ 517 17,670-+ 383
Escherichia coli* Salmonella typhimurium* Salmonella enteriditis*
Cultures were pulsed with 3H-Tdr of SA 370 mCi/mM. *These endotoxins were prepared by the method of Westphal et al. (1952), and were a gift from Dr. W.H. Adler.
396
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Mitogen dilution (10tal added per culture)
Fig. 7. The effect of mitogen dose upon the 3H-Tdr incorporation of mouse lymphocytes: (a) Stimulation by Con A and LPS: o. . . . . o = actual incorporation obtained with LPS using 3H-Tdr of SA 370 mCi/mM; o o = calculated incorporation obtained with LPS using 3H-Tdr of SA 150 mCi/mM to facilitate comparison with the other mitogens; (b) Stimulation with PHA and PWM. Mitogen dilution refers to the dilution of the 5 ml reconstituted material. Pulsed with 3H-Tdr of SA 150 mCi/mM (except o. . . . . o).
3.9. Effect of culture time The optimal culture time is dependent upon the initial cell concentration, higher cell concentrations favouring shorter culture times. This effect is shown for Con A in fig. 8a. The optimal culture period for Con A, PHA and PWM was 72 hr at the optimal cell concentration of 6 × 10 s cells per culture. For LPS, the optimal culture period was 24 hr earlier at 48 hr (fig. 8b). The magnitude of the undirectional MLC was less dependent upon the culture time than were the mitogen responses (fig. 9). As was apparent in the mitogen studies, a higher cell concentration favoured an earlier optimal culture period for MLC, although this relation was less marked. Maximal response in undirectional MLC was obtained between 4 8 - 7 2 hr of culture, with 1 to 1.5 X 106 responding cells per culture.
3.10. Preliminary comparison of mitogen responses of mesenteric lymph node, peripheral lymph nodes and spleen Cell suspensions were prepared from pooled mesenteric nodes, spleens, and from inguinal and axial nodes cultured under the optimal conditions as determined for mesenteric lymphocytes. Results are given in table 3. Splenic lymphocytes showed a greater responsiveness to LPS and PWM than either mesenteric or peripheral lymph node lymphocytes, whereas the responsiveness to PHA or Con A was not markedly different from lymph node lymphocytes. Unstimulated control values for
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Culture time (hours) Fig. 8..Effect of culture time on 3H-Tdr incorporation of mouse lymphocytes in response to mitogens: (a) Inter-relationship of optimal culture time and initial cell concentration for stimulation with Con A. A A = unstimulated control; (b) Stimulation with PHA, LPS and PWM. o . . . . . . o = actual incorporation obtained with LPS using 3H-Tdr of SA 370 mCi/mM. o--o = calculated incorporation obtained with LPS using 3H-Tdr of SA 150 mCi/mM to facilitate comparison with the other mitogens. A A = unstimulated control for PHA and PWM stimulation, v v = unstimulated control for LPS stimulation. Pulsed with 3H-Tdr of SA 150 mCi/mM (except o . . . . . -o).
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Culture time (hours) Fig. 9. Effect of culture time on 3H-Tdr incorporation of mouse lymphocytes in response to unidirectional MLC. n D = response of 1.0 × 106 CBA lymphocytes per culture to 0.5 X 106 mitomycin-C-treated C57 BL/10 lymphocytes per culture, a ~ = syngeneic mixture of 1.0 X 106 CBA lymphocytes per culture and 0.5 X 106 mitomycin-C-treated lymphocytes per culture. Pulsed with 3 H-Tdr of SA 370 mCi/mM.
398
P.E. THORPE and S.C. KNIGHT
Table 3 Comparison of 3H-Tdr incorporation in response to mitogens in lymphocyte cultures derived from mesenteric lymph node, peripheral lymph node and spleen. Mitogen
None PHA Con A PWM LPS
3H-Tdr incorporation (mean dpm ± S.E.) Mesenteric lymph node
Peripheral lymph nodes
Spleen
1395 101,720 107,110 10,514 14,450
3287 102,210 139,170 16,950 11,630
8875 111,450 128,030 22,400 35,040
± 110 ± 3930 ± 3160 ± 870 ± 550
± 290 -+4920 ± 4070 ± 850 ± 930
± 545 ± 6460 ± 4090 ± 2590 ± 3290
Cultures were pulsed with 3H-Tdr of SA 370 mCi/mM.
splenic lymphocytes were considerably greater than for lymph node lymphocytes. The responses o f peripheral lymph node lymphocytes were fairly similar to those of mesenteric lymphocytes, the primary difference being in the 2-fold greater control value of the peripherally derived cells.
3.11. Effect o f concentration o f exogenous 3H- Tdr Ten microliters of 3H-Tdr solutions of varying concentrations from 5 to 160 pg/rnl, and o f constant low SA (150 mCi/mM) was added to identical cultures stimulated optimally with PHA, Con A or PWM. Cultures were harvested 24 hr later. Flooding conditions of exogenous 3H.Tdr were achieved for a 24 hr-pulse with 10/al/culture of Tdr solution o f concentration greater than 60/lg/ml (0.6/ag/ culture) for stimulation with Con A or PHA, and at greater than 30/ug/ml (0.3 ~ug/ culture) for stimulation with PWM (fig. 10). There was no indication that the Tdr concentration reached inhibitory levels at 1.6/ag/culture.
3.12. Effect o f specific activity o f 3H- Tdr Twenty microliters of aH.Tdr solutions of constant total Tdr concentration (27.5/ag/ml) and varying SA from 24 to 880 mCi/mM was added to identical cultures stimulated optimally with PHA, Con A or PWM. Cultures were harvested 24 hr later and results are shown in fig. 11. The relationship between SA and 3 H-Tdr incorporation is paraboloid. The mathematical equations of the parabolas obtained for PHA, Con A and PWM-stimulated cultures, and for unstimulated control cultures, were calculated from each individual value in fig. 11, and were seen to be mathematically similar. The SA that allows maximal a H-Tdr incorporation under saturation conditions with a 24 hr-pulse was calculated to be 1.3 Ci/mM. A SA o f 370 mCi/mM was chosen to enable easily measureable dpm to be obtained with weak stimulants.
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399
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400
P.E. THORPE and S.C. KNIGHT
4. DISCUSSION The relationship between cell concentration in culture and proliferation in response to mitogens is influenced by such factors as availability of nutrients and mitogens, fall in pH and cell interactions (Ling, 1968). As these factors affect cultures with each stimulant, the differences in the relationship between cell concentration and proliferation for each stimulant probably reflect differences in the populations of cells being activated, and their mode of activation. Thus, the marked similarity in the kinetics of lymphocyte responses to Con A and PHA may indicate that they activate similar populations of cells by similar mechanisms. The proliferation in cultures stimulated with PWM is a plateau over a wide range of cell concentrations (compared with responses to Con A, LPS and optimal and suboptimal doses of PHA) possibly because PWM stimulates both T and B cells and effectively combines the Con A or PHA and the LPS response curves. This provides suggestive evidence that PWM causes independent stimulation of T and B cells. The importance of the shape of the culture vessel has been stressed previously for tube cultures (Moorhead et al., 1967; Ling, 1968) and for microplate cultures (Thurman et al., 1973). Round-bottomed wells were shown in this study to allow optimal stimulation at a lower cell concentration than flat-bottomed wells. It is probable that the choice of culture vessel affects the kinetics of the MLC response to a greater extent than mitogen responses due to the more conspicuous requirement for cell-cell contact (Chapman and Dutton, 1965; Moorhead et al., 1967). Hence, by comparison with mitogen studies, previous reports on the kinetics of the MLC response show greater variance (Adler et al., 1970; Mangi and Mardiney, 1970; Phillips et al., 1972; Peck and Bach, 1973). Consistently low PWM responses were found in this study compared with those obtained by other workers (Janossy and Greaves, 1971; Strong et al., 1973), and even under optimal conditions these only reached 10% of the PHA response. Lymphocytes from spleen and peripheral lymph nodes gave improved PWM responses compared to mesenteric lymphocytes, but these were still low at 21% and 16% of the PHA responses respectively. The mice used in this study were CBA female mice which have been reported to give considerably lower responses to PWM compared to CBA male mice (Strong et al., 1973). In humans, lymphocytes from males and females give similar PWM responses (unpublished observations). The log dose-response curves for Con A, PHA, PWM and LPS show approximately linear responses at dose levels below the optimum. For Con A and PHA, excess mitogen appears to be tolerogenic, since elution of the mitogen can enable recovery of the full response (Andersson et al., 1972b; Knight and Farrant, unpublished observations). Both LPS and PWM show a marked lack of either tolerogenicity or toxicity as shown by the very broad log dose-response curves. The optimal culture period for cells activated by a particular stimulant is probably determined by such factors as the characteristics of the target cell, the celt concentration and the magnitude of stimulation. Therefore, the earlier response to
Microplate culture o f mouse lymph node cells
401
LPS compared to Con A or PHA, may be a characteristic of the B-cell target, although PWM at a cell concentration of 3 × 106/ml does not show this earlier peak, or may merely be a manifestation of the high cell concentration required in LPS culture. Mitomycin-C treatment as a means of inactivating the stimulating population for unidirectional MLC allowed in a greater degree of activation, and at a lower optimal cell concentration that did irradiation. This was probably because cell death due to irradiation damage, although not initially obvious from trypan blue dye exclusion counts, occurred early during culture (Schrek, 1959). For lymphocyte culture, two buffers fulfil the necessary requirements of a pKa close to the optimum pH of the system, and low toxicity. These are sodium bicarbonate (pKa = 6.10 at 37°C, requiring equilibration with CO2 for maintenance of pH) and the sulphonic acid derivative HEPES (pKa = 7.31 at 37°C, Good et al., 1966). The buffering capacity of HEPES (20 raM) at the optimal culture pH of 7.34 is 1.1 X 10-2 g. equiv/pH unit compared to 0.27 X 10-2 g. equiv./pH unit for bicarbonate buffer (25 mM). These studies showed that bicarbonate buffer allowed at least 50% greater stimulation of mouse lymphocytes by mitogens than did HEPES buffer. Manipulation of the cells in HEPES-buffered medium before transfer to bicarbonate-buffered medium for culture overcame the gassing problem during handling, and allowed a further increase in stimulation. The stimulation obtained with HEPES buffer could not be improved by adjustment of HEPES concentration or initial pH. The small improvement in stimulation achieved upon addition of 5 mM bicarbonate approximated to the calculated increment allowed by the resulting mixed buffer system. Therefore, the inferiority of HEPES buffer compared to bicarbonate buffer was not due to an inferior buffering ability or to deficiency of essential bicarbonate ion. The possibility that HEPES was toxic to mouse lymphocytes was supported by the observation that even with low stimulation levels, such as with PWM and suboptimal PHA, when the demand for pH control is small HEPES was inferior to bicarbonate to the same extent as at high stimulation levels. Moreover, the small effect of the HEPES concentration upon cell proliferation (fig. 4) in view of the critical nature of the pH of culture (fig. 2) may indicate the interplay of an advantageous pH control effect and a disadvantageous effect of toxicity. Human lymphocytes responding to mitogen or MLC have been shown to allow equivalent stimulation with HEPES and bicarbonate buffers (Darzynkiewicz and Jacobson, 1971; Thurman et al., 1973)and Eagle (1971)showed that HEPES (20 raM) in combination with bicarbonate (24 raM) was not toxic to several mammalian cell types. Presumably mouse lymphocytes are unusually sensitive to HEPES toxicity. The optimal initial pH was dependent upon the initial cell concentration for cells stimulated with Con A, a higher cell concentration requiring a more alkaline pH. This is presumably because a greater cell number has a higher acid metabolite production, therefore an alkaline pH effectively neutralises the developing acidity. This is supported by preliminary data that indicates that addition of alkali to
402
P.E. THORPEand S.C. KNIGHT
cultures after 24 hr, as opposed to an initial change in pH, further increases stimulation in response to Con A in cultures of high cell concentration (1.2 × 106 per culture). The diminution of 3 H-Tdr uptake in Con A or PHA-stimulated cultures that is seen with cell concentration greater than 6 × l0 s cells per culture is, therefore, due, at least in part, to the fall in pH. The degree of activation of lymphocytes is assessed from the rate of DNA synthesis, as monitored by the incorporation of 3 H-Tdr. The concentration and SA of exogenous 3H-Tdr and the pulse time affect the correlation between rate of incorporation of label and rate of DNA synthesis (Cleaver, 1967). The pulse time of 24 hr was maintained throughout this study because the incorporation of label approaches linearity with pulse times of up to 24 hours at flooding concentrations of exogenous Tdr. (Schellekens and Eijsvoogel, 1968; Bain, 1970' Shons et al., 1972). This enables good incorporation of label to be achieved with 3 H-Tdr of low SA. The exogenous 3H_Tdr diffuses into the cell where it is phosphorylated to the mono-, di- or triphosphonucleotides (Bucher, 1963). The SA of these products is reduced by the dilutional effect of endogenous Tdr nucleotides. The incorporation of label into the DNA depends upon the resultant SA of the intracellular pool of Tdr triphosphate, and is maximal under conditions of excess exogenous Tdr. Under such saturation conditions the SA of the intracellular pool is constant during the 3 H-Tdr pulse, the % incorporation of label is small and volumetric errors during addition of 3H.Tdr solution are minimised. The closest correlation is therefore achieved between rate of incorporation of label and rate of DNA synthesis (Sample and Chretien, 1971 ; Quastler, 1963; Cleaver, 1967). In these studies, flooding conditions were achieved for a 24 hr-pulse with exogenous Tdr concentrations of 3.0/~g/ml in culture for optimal Con A and PHA stimulation. For lower levels of activation the % incorporation of exogenous Tdr is less, and flooding conditions are achieved with lower Tdr concentrations. At flooding Tdr concentrations, the SA of the intracellular pool ofTdr nucleotides is directly related to the SA of the exogenous 3 H-Tdr. However, the relationship between incorporation of label into DNA and the SA departs from linearity due to cellular inactivation from radiation damage and the percentage departure from linearity is proportional to the SA. Hence the rate of change of gradient in a graph of 3 H-Tdr incorporation versus SA is a constant, and double integration of this relationship gives the equation of a parabola. Mathematical treatment of each individual value for the dpm versus SA curves in fig. 11 enables the elaboration of the equations for each curve. The parabolic changes of dpm with SA for Con A, PHA and PWM and unstimulated control values are mathematically similar in spite of the markedly different degrees of activation. It is valid, therefore, for quantitation of the response to use any SA of 3 H-Tdr depending upon expense and required dpm, providing that flooding concentrations of Tdr are maintained throughout the pulse. The SA of 3 H-Tdr that gives maximal dpm with a 24 hr-pulse with saturation concentrations of Tdr can be calculated from fig. 11 to be 1.3 Ci/mM, and at
Microplate culture of mouse lymph node cells
403
greater SA the calculated dpm diminish. It is likely that the cellular inactivation due to the radiobiological effects is proportional to the pulse time, therefore for shorter pulses than 24 hr, the maximal SA can be increased proportionately.
ACKNOWLEDGEMENTS We gratefully acknowledge the help and discussion of Sir Peter Medawar, Dr. Noel Ling and Dr. Colin Sanderson. This work was supported by a grant from the Medical Research Council. REFERENCES Adler, W.H., T. Takiguchi, B. Marsh and R.T. Smith, 1970, J. Immunol. 105,984. Andersson, J., G. Mtiller and O. Sj6berg, 1972a, Cell. Immunol. 4, 381. Andersson, J., O. Sjoberg and G. MiSller, 1972b, Immunology 23,637. Bain, B., 1970, Clin. Exptl. Immunol. 6,255. Brody, J.A. and B. Huntley, 1965, Nature 208, 1232. Bucher, N.L.R., 1963, Intern. Rev. Cytol. 15,245. Carr, M.C., D.P. Stites and H.H. Fudenberg, 1973, Nature New Biol. 241,279. Chapman, N.D. and R.W. Dutton, 1965, J. Exptl. Med. 121,85. Cleaver, J.E., 1967, in: Thymidine metabolism and cell kinetics, eds. A. Neuberger and E.L. Tatum, (North Holland, Amsterdam.) Darzynkiewicz, Z. and B. Jacobson, 1971, Proc. Soc. Exptl. Biol. Med. 136,387. Davies, A.J.S., H. Festenstein, E. Leuchars, V.J. Wallisand M.J. Doenhoff, 1968, Lancet I, 183. Diamond, B., S.C. Knight and E.M. Lance, 1974, Cell. Immunol. II, 239. Eagle, H., 1971, Science 174,500. Gery, I., J. Kr'tigerand S.Z. Spiesel, 1972, J. Immunol. 108, 1088. Good, N.E., G.D. Winget, W. Winter, T.N. Connolly, S. Izawa and R.M.M. Singh, 1966, Biochemistry 5,467. Janossy, G., and M.F. Greaves, 1971, Clin. Exptl. Immunol. 9,483. Janossy, G. and M.F. Greaves, 1972, Clin. Exptl. Immunol. 10, 525. Knight, S.C., B. Newey and N.R. Ling, 1973a, Cytobios 7, 35. Knight, S.C., B. Newey and N.R. Ling, 1973b, Cell. Immunol. 9,273. Ling, N.R., 1968, in: Lymphocyte Stimulation (North Holland, Amsterdam). Mangi, R.J. and M.R. Mardiney, 1970, J. Immunol. 105, 90. Moorhead, J.F., J.J. Connally and W. McFarland, 1967, J. Immunol. 99,413. Parker, J.W. and R.J. Lukes, 1971, Amer. J. Clin. Pathol. 56,174. Peck, A.B. and F.H. Bach, 1973, J. Immunol. Methods 3,147. Phillips, S.M., C.B. Carpenter and J.P. Merrill, 1972, Cell. Immunol. 5,235. Quastler, H., 1963, in: Actions Chimiques et Biologiques des Radiations, ed. M. Haissinsky (Masson et Cie, Paris). Rieke, W.O. and M.R. Schwarz, 1967, Acta Haemat. 38,121. Sample, W.F. and P.B. Chretien, 1971, Clin. Exptl. Immunol. 9,419. Schellekens, P.Th.A. and V.P. Eijsvoogel, 1968, Clin. Exptl. Immunol. 3, 571. Schrek, R., 1959, Am. J. Clin. Pathol. 31,112. Schrek, R. and K.V. Batra, 1966, Lancet II, 444. Schwarz, M.R., 1967, Amer. J. Anat. 121,559.
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Shons, A.R., E.E. Etheredge and J.S. Najarian, 1972, Clin. Exptl. Immunol. 12,351. Stobo, J.D. and W.E. Paul, 1973, J. Immunol. 110, 362. Strong, D.M., A.A. Ahmed, G.B. Thurman and K.W. Sell, 1973, J. lmmunol. Methods 2,279. Thurman, G.B., D.M. Strong, A. Ahmed, S.S. Green, K.W. Sell, R.J. Hartzman and F.H. Bach, 1973, Clin. Exptl. Immunol. 15,289. Westphal, O., Liideritz and F. Bister, 1952, Z. Naturforsch Teil. B. 7b, 148. Wilson, D.B., W.K. Silvers and P.C. Nowell, 1967, J. Exptl. Med. 126,655.