Optimal Macroculture Method for Studying Mitogenic Stimulation of Turkey Lymphocytes J. Tessler and L. A. Page* ABSTRACT
This report describes a method for culturing turkey lymphocytes in disposable, unwashed glass test tubes with Morton closures and for recovering lymphocytes on fiber glass filters with a cell harvester made of common laboratory equipment for assay of mitogenic stimulation. Optimal conditions for culture were established.
RASUME Les auteurs decrivent une methode de cultiver des lymphocytes de dindes dans des eprouvettes en verre, non lavees, utilisables qu'une seule fois et pourvues de bouchons de Morton; ils donnent aussi la fagon de recouvrer les lymphocytes sur des filtres en fibre de verre, a l'aide d'un recolteur de cellules constitue d'equipement conventionnel de laboratoire destine a experimenter la stimulation mitotique. ls enumerent en dernier lieu les conditions optimales de la culture des lymphocytes.
Determination of the degree of blastogenesis of lymphocytes cultured in the presence of various mitogens is a widely applied tool in immunology, and a variety of methods for culturing and recovering tissue cells for mitogenic analysis have been published for mammals and fowls other *National Animal Disease Center, Science and Education Administration, U.S. Department of Agriculture, P.O. Box 70, Ames, Iowa 50010. No endorsements implied herein. Submitted September 8, 1977.
Volume 42 - April, 1978
than the turkey (1, 2, 4, 5, 6). Most of the methods required the use of expensive multiple cell harvesters. Sophisticated equipment for microculture and rapid mechanical harvest of cultured lymphocytes was not available and only common laboratory glassware and supplies were used to perform the tests. This report describes optimal conditions necessary to prepare and culture turkey lymphocytes in disposable glass test tubes with Morton closures or with capped plastic test tubes and the use of a simple cell harvester (2) for collecting incubated lymphocytes on fiber glass filters for final mitogenic assay. Broad-breasted white turkeys eight to 15 weeks old were used. They were obtained as day-old poults from a commercial source and raised in isolation quarters with controlled entry and exit of air and water. Peripheral lymphocytes from the turkeys were cultured in RPMI 16401 with varied concentrations of fetal calf serum (FCS) prepared at the National Animal Disease Center and with varied concentrations of phytohemagglutinin (PHA-P).2 A less pure mitogen, Difco PHA-M,3 was used earlier and rejected because it induced very low mitogenic responses with turkey lymphocytes. Benzylpenicillin potassium (100 units/ml) and streptomycin sesqui sulfate (50 ug/ml) were added to all culture media. In early experiments, lymphocytes purified by the ficoll-diatrizoate technique of B6yum (3) failed to undergo blastogenesis in the presence of active mitogens. Therefore, our final protocol was a modification lGrand Island Biological Company, Grand Island, New York.
2Difco Laboratories, Detroit, Michigan. 3Difco Laboratories, Detroit, Michigan.
249
of the method of Kirchner (5). Specifically, 4.5 ml of blood was drawn from turkeys into a syringe containing 0.5 ml of heparin solution (500 units/ml), introduced into a centrifuge tube and immediately centrifuged at 120 x G for three minutes. Lymphocyte-rich supernatant fluid and buffy coat free of red blood cells were withdrawn with a mechanical pipetter (PiPump)4 and washed twice for two minutes at 275 x G in culture medium containing 15% FCS and two units/ml of heparin. The cells were resuspended in five ml of medium without heparin and counted in a hemacytometer by staining the lymphocytes with the Natt-Herrick method (8). Varying concentrations of lymphocytes contained in 0.4 ml of medium were dispensed into sterile disposable polystyrene tubes (12 x 75 mm) with caps or disposable, unwashed glass serological tubes (13 x 100 mm) capped with Morton closures5 (7) that had been sterilized by autoclaving. In experiment five, lymphocytes were cultured in multi-dish "disposotrays"I with 16 mm diameter flat-bottom wells. Medium (RPMI 1640) containing different concentrations of PHA-P were added in 0.2 ml amounts to each tube. Control cultures contained medium alone. In preliminary trials, lymphocyte cultures were incubated at 37°C for 50-72 hours in an atmosphere of 5% C02 and 100% relative humidity followed by a 16-hour labeling with 3H-thymidine. Because there was no advantage for the longer incubation triplicate cultures of each sample, with and without mitogen, were incubated at 370C for 50 hours in the final protocol. One juCi of 3H-thymidine (6.7 curies/ ,umole)7 in 0.05 ml of medium was then added to each tube culture. Incubation was continued for 16 hours at 37°C, then the cultures were harvested. In early experiments, greater mitogenic responses in terms of stimulation index (SI) were derived in cultures from which supernatant fluids were carefully decanted and discarded just before harvest. The harvest procedure after this step was as follows. Five ml of distilled water was pipetted into each tube with a Cornwall 4Bel-Art Products, Pequannock, New Jersey. 5Bellco Laboratories, Vineland, New Jersey. 6Linbro Chem Company, Inc., New Haven, Connecticut. 7New England Nuclear Corp., Boston, Massachusetts.
250
automatic syringe to resuspend and lyse the cells. This suspension was decanted into a 5 ml open syringe barrel attached to a Swinnex8 filter holder containing a 13 mm diameter fiber glass filter (Whatman GF/C)9 which in turn was attached to a vacuum box and pump. This apparatus was designed by Alhajii et al for bovine lymphocytes (2). The culture tubes were then washed twice with 2.5 ml of distilled water, each aliquot of which was decanted into the syringe. After all the cultures were harvested, the filters were removed and placed in proper sequence in numbered glass scintillation vials and dried in a vacuum oven for 60 minutes at 121°C. Ten milliliters of PPO-POPOP scintillant (0.5% 2,5-diphenyloxazole and 0.03% 2,2' (1,4-phenylene) bis (4-methyl-5-phenyl oxazolyl) in toluene) was added to each vial. The rate of tritium decay over a period of five minutes was determined in a scintillation counter (Packard Tri-Carb Model 3380). The uptake of 3H-thymidine was expressed as net counts per min (CPM) derived from the mean value of triplicate determinations. A lymphocyte SI for cultures incubated with PHA-P was calculated by dividing the average net count of cultures without PHA-P into average net counts of cultures with PHA-P. When the effect of adding fetal calf serum to the lymphocyte cultures was tested, 3H-thymidine uptake in the presence of PHA-P was somewhat higher when FCS was incorporated at a concentration of 10% than when FCS was left out (Table I). When the concentration of FCS was raised to 15% in later experiments (Table IV) a further beneficial effect on lymphocytic blastogenesis (as measured by 3Hthymidine) was observed. This result is in contrast to the work of Maheswaran et al (6). We did not obtain mitogen-stimulated blastogenesis of turkey lymphocytes in the absence of serum in the culture medium. There were some differences in concentration of lymphocytes/ml of culture used in Maheswaran's and our tests. Maheswaran used 20 x 106 lymphocytes/ml of culture (6) and we used 2.1 x 100 lymphocytes/ml. The larger number of cells used by 8Millipore Corporation, Bedford, Massachusetts. 9Reeve-Angel Corporation, Clifton, New Jersey. 1OPackard-Hewlett Corporation, Downers Grove, Illinoi.
Can. J. comp. Med.
TABLE I. Effect of Addition of Fetal Calf Serum on Blastogenic Stimulation of Turkey No. 3033 Lymphocytes by Phytohemagglutinin with Polystyrene Tubes
Exp. No. 1
10% Fetal CallIf Serum in Cultures No Serum in Cultures Culture 3H-Thymidine Uptake Stimulation 3H-Thymidine bUptake Stimulation Index CPM CPMb Additionsa Index 200 4,070 2.5 PHA-P,o 13 j1 15.9 256 79 None
852 5.5 19.738 PHA-P, 13 pd 1,059 154 None 11,424 1.4 1,529 3 PHA-P, 13 ,L 936 1,116 None aO.62 x 106 lymphocytes/culture in 0.3 ml bCPM, counts per minute ,Stock solution of phytohemagglutinin (Difco, purified) in 5 ml of culture medium
18.6
2
12.2
TABLE II. Effect of Doubling Culture Volume and Contents and Varying Culture Containers on Blastogenic Stimulation of Turkey No. 3030 Lymphocytes by Phytohemagglutinin
Exp. No. 4
0.62 x 106 Lymphocytes in 0.3 ml of 1.25 x 106 Lymphocytes in 0.6 ml of Medium with 13 wl PHA-P and 10% Medium with 26 [LI PHA-P and 10% FCS FCSb Culture 3H-Thymidine Uptake Stimulation 3H-Thymidine Uptake Stimulation CPM Index Index CPMc Additionsa 13 x 100 mm Glass Test Tubes
2,014 298
PHA-P None
6.7
27.0
10,656 394
Plastic Flat Bottom Culture Plates
2.5 PHA-P 900 3,589 856 362 None RPHA-P, phytohemagglutinin (Difco, purified) In 5 ml of culture medium bFCS, fetal calf serum °CPM, counts per minute
4.2
5
TABLE III. Effects of Decantation of Culture Fluid before Turkey No. 3031 Lymphocyte Harvest and Lymphocyte Concentration on Stimulation of Blastogenesis by Phytohemagglutinin In Glass Tubes (Experiment 6)
No. Lymphocytes per 0.6 ml Culture 0.62 x 106
Culture Additionsa PHA-P,° 13 Al None
Decanted Culture Supernatant
Nondecanted Culture Supernatant
3H-Thymidine Stimulation Uptake CPMb Index 56.1 8,356 149 190.8 13,929
3H-Thymidine Stimulation Uptake
PHA-P, 26 I.l 73 None 149.3 2.5 x 106 14,187 PHA-P, 39 p1 95 None 43.0 5,038 3.75 x 106 PHA-P, 52 p.l 117 None -All cultures contained 10% fetal calf serum bCPM, counts per minute °PHA-P, phytohemagglutinin (Difco, purified) in 5 ml culture medium 1.25 x 106
Volume 42
-
April, 1978
11,930
Index 71.9
15,739
64.2
15,454
48.3
11,480
29.0
CPM
166
245
320
396
251
TABLE IV. Average Indices of PHA-Stimulated Blastogenesis of Turkey Lymphocytes Cultured in Glass Test Tubes with Morton Closures
Exp. No. 7
Conc. FCSb in Culture Medium 10%
Average for Ten Adult Turkeys
3H-Thymidine Uptake CPM° (range) 2,944 (2,1884,319) 444 (250-737) 10% 2,870 (1,371-5,217) 408 (344-648) 15% 11,079 (4,470-27,850) 526 (326-718) 15% 13,250 (2,900-20,981) 761 (507-1,648) 15% 12,598 (1,30944,830) 343 (224-353) 15% 10,446 (3,836-24,676) 251 (180-449) 1.25 x 106 lymphocytes in 0.6 ml
Culture Additionsa PHA-Pd None
Stimulation Index (range)e 6.6 (4.9-8.9)
PHA-P 7.0 (3.2-8.9) None PHA-P 9 21.1 (8.5-36) None PHA-P 10 17.4 (5.9-36) None PHA-P 11 36.7 (4.7-131) None PHA-P 12 41.6 (12.1-78) None -All cultures contained bFCS, fetal calf serum CCPM, counts per minute dPHA-P, phytohemagglutinin (Difco, purified) in 5 ml culture medium, 26 ul was added to each culture eThis figure is the average of all individual SI determinations
8
Maheswaran (6) may not have required serum as suggested by Kirchner and Oppenheim (5). Doubling the total culture volumes (including lymphocytes and PHA-P) from 0.3 to 0.6 ml also increased blastogenic stimulation (Table: II). Decanting and discarding culture supernatant fluids just before harvest of incubated lymphocytes also increased SI's (Table III). Removal of the supernatant fluids without disturbing the lymphocytes settled on the bottom of the culture tube eliminated 3H-thymidine that was unincorporated in lymphocyte DNA. This lowered detectable CPM's in control cultures and thereby ultimately raised the computed SI. Incorporating all of the above modifications, an average SI of 29.2 was obtained in cultures containing PHA-P and 1.25 x 106 lymphocytes in 0.6 ml of medium with 15% FCS in glass tubes with Morton closures (Table IV). This protocol was used to monitor lymphocyte blastogenesis stimulated by antigens of Chlamydita psittaci in turkeys vaccinated with chlamydial bacterins (L. A. Page and J. Tessler, Abstracts of Annual Meeting of American Society for Microbiology, Atlantic City, New Jersey 1976, Abst. E-135 and L. A. Page, Am. J. Vet. Res. 39: 473-480. 1978).
252
ACKNOWLEDGMENTS We thank Drs. C. C. Muscoplat and D. W. Johnson of the University of Minnesota for valuable advice and loan of cell harvesting equipment used throughout this experimentation and Mr. Joseph Wells for his technical assistance.
REFERENCES 1. ADLER, W. H., T. TAKIGUCHI, B. MARSH and R. T. SMITH. Cellular recognition by mouse lymphocytes in vitro. I. Definition of a new technique and results of stimulation by phytohemagglutinin and specific antigens. J. exp. Med. 131: 1049-1078. 1970. 2. ALHAJII, I., D. W. JOHNSON, C. C. MUSCOPLAT and C. 0. THOEN. Diagnosis of Mycobacterium bovis and MYcobacterium paratuberculosis infections in cattle by in vitro lymphocyte immunostimulation. Am. J. vet. Res. 35: 725-727. 1974. 3. BOYUM, A. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. clin. lab. Invest. 21 (suppl. 97): 77-89. 1968. 4. FERNALD, G. W. and R. S. METZGAR. In vitro studies of the response of hamster lymphocytes to phytohemagglutinin and antigenic stimulation. J. Immun. 107: 456-463. 1971. 5. KIRCHNER, H. and J. J. OPPENHEIM. Stimulation of chicken lymphocytes in serum-free medium. Cell. Immunol. 3: 695-699. 1972. 6. MAHESWARAN, S. K., E. S. THIES and C. GREIMANN. A micromethod for evaluating turkey lymphocyte responses to phytomitogens. Am. J. vet. Res. 36: 1397-1400. 1975. 7. MORTON, H. E. Stainless steel culture tube closures as a replacement for cotton plugs. Science 126: 1248. 1957. 8. NATT, M. P. and C. A. HERRICK. A new blood diluent for counting the erythrocytes and leucocytes of the chickens. Poult. Sci. 3: 735-738. 1952.
Can. J. comp. Med.
This report describes a method for culturing turkey lymphocytes in disposable, unwashed glass test tubes with Morton closures and for recovering lymphocytes on fiber glass filters with a cell harvester made of common laboratory equipment for assay of mitogenic stimulation. Optimal conditions for culture were established.
RASUME Les auteurs decrivent une methode de cultiver des lymphocytes de dindes dans des eprouvettes en verre, non lavees, utilisables qu'une seule fois et pourvues de bouchons de Morton; ils donnent aussi la fagon de recouvrer les lymphocytes sur des filtres en fibre de verre, a l'aide d'un recolteur de cellules constitue d'equipement conventionnel de laboratoire destine a experimenter la stimulation mitotique. ls enumerent en dernier lieu les conditions optimales de la culture des lymphocytes.
Determination of the degree of blastogenesis of lymphocytes cultured in the presence of various mitogens is a widely applied tool in immunology, and a variety of methods for culturing and recovering tissue cells for mitogenic analysis have been published for mammals and fowls other *National Animal Disease Center, Science and Education Administration, U.S. Department of Agriculture, P.O. Box 70, Ames, Iowa 50010. No endorsements implied herein. Submitted September 8, 1977.
Volume 42 - April, 1978
than the turkey (1, 2, 4, 5, 6). Most of the methods required the use of expensive multiple cell harvesters. Sophisticated equipment for microculture and rapid mechanical harvest of cultured lymphocytes was not available and only common laboratory glassware and supplies were used to perform the tests. This report describes optimal conditions necessary to prepare and culture turkey lymphocytes in disposable glass test tubes with Morton closures or with capped plastic test tubes and the use of a simple cell harvester (2) for collecting incubated lymphocytes on fiber glass filters for final mitogenic assay. Broad-breasted white turkeys eight to 15 weeks old were used. They were obtained as day-old poults from a commercial source and raised in isolation quarters with controlled entry and exit of air and water. Peripheral lymphocytes from the turkeys were cultured in RPMI 16401 with varied concentrations of fetal calf serum (FCS) prepared at the National Animal Disease Center and with varied concentrations of phytohemagglutinin (PHA-P).2 A less pure mitogen, Difco PHA-M,3 was used earlier and rejected because it induced very low mitogenic responses with turkey lymphocytes. Benzylpenicillin potassium (100 units/ml) and streptomycin sesqui sulfate (50 ug/ml) were added to all culture media. In early experiments, lymphocytes purified by the ficoll-diatrizoate technique of B6yum (3) failed to undergo blastogenesis in the presence of active mitogens. Therefore, our final protocol was a modification lGrand Island Biological Company, Grand Island, New York.
2Difco Laboratories, Detroit, Michigan. 3Difco Laboratories, Detroit, Michigan.
249
of the method of Kirchner (5). Specifically, 4.5 ml of blood was drawn from turkeys into a syringe containing 0.5 ml of heparin solution (500 units/ml), introduced into a centrifuge tube and immediately centrifuged at 120 x G for three minutes. Lymphocyte-rich supernatant fluid and buffy coat free of red blood cells were withdrawn with a mechanical pipetter (PiPump)4 and washed twice for two minutes at 275 x G in culture medium containing 15% FCS and two units/ml of heparin. The cells were resuspended in five ml of medium without heparin and counted in a hemacytometer by staining the lymphocytes with the Natt-Herrick method (8). Varying concentrations of lymphocytes contained in 0.4 ml of medium were dispensed into sterile disposable polystyrene tubes (12 x 75 mm) with caps or disposable, unwashed glass serological tubes (13 x 100 mm) capped with Morton closures5 (7) that had been sterilized by autoclaving. In experiment five, lymphocytes were cultured in multi-dish "disposotrays"I with 16 mm diameter flat-bottom wells. Medium (RPMI 1640) containing different concentrations of PHA-P were added in 0.2 ml amounts to each tube. Control cultures contained medium alone. In preliminary trials, lymphocyte cultures were incubated at 37°C for 50-72 hours in an atmosphere of 5% C02 and 100% relative humidity followed by a 16-hour labeling with 3H-thymidine. Because there was no advantage for the longer incubation triplicate cultures of each sample, with and without mitogen, were incubated at 370C for 50 hours in the final protocol. One juCi of 3H-thymidine (6.7 curies/ ,umole)7 in 0.05 ml of medium was then added to each tube culture. Incubation was continued for 16 hours at 37°C, then the cultures were harvested. In early experiments, greater mitogenic responses in terms of stimulation index (SI) were derived in cultures from which supernatant fluids were carefully decanted and discarded just before harvest. The harvest procedure after this step was as follows. Five ml of distilled water was pipetted into each tube with a Cornwall 4Bel-Art Products, Pequannock, New Jersey. 5Bellco Laboratories, Vineland, New Jersey. 6Linbro Chem Company, Inc., New Haven, Connecticut. 7New England Nuclear Corp., Boston, Massachusetts.
250
automatic syringe to resuspend and lyse the cells. This suspension was decanted into a 5 ml open syringe barrel attached to a Swinnex8 filter holder containing a 13 mm diameter fiber glass filter (Whatman GF/C)9 which in turn was attached to a vacuum box and pump. This apparatus was designed by Alhajii et al for bovine lymphocytes (2). The culture tubes were then washed twice with 2.5 ml of distilled water, each aliquot of which was decanted into the syringe. After all the cultures were harvested, the filters were removed and placed in proper sequence in numbered glass scintillation vials and dried in a vacuum oven for 60 minutes at 121°C. Ten milliliters of PPO-POPOP scintillant (0.5% 2,5-diphenyloxazole and 0.03% 2,2' (1,4-phenylene) bis (4-methyl-5-phenyl oxazolyl) in toluene) was added to each vial. The rate of tritium decay over a period of five minutes was determined in a scintillation counter (Packard Tri-Carb Model 3380). The uptake of 3H-thymidine was expressed as net counts per min (CPM) derived from the mean value of triplicate determinations. A lymphocyte SI for cultures incubated with PHA-P was calculated by dividing the average net count of cultures without PHA-P into average net counts of cultures with PHA-P. When the effect of adding fetal calf serum to the lymphocyte cultures was tested, 3H-thymidine uptake in the presence of PHA-P was somewhat higher when FCS was incorporated at a concentration of 10% than when FCS was left out (Table I). When the concentration of FCS was raised to 15% in later experiments (Table IV) a further beneficial effect on lymphocytic blastogenesis (as measured by 3Hthymidine) was observed. This result is in contrast to the work of Maheswaran et al (6). We did not obtain mitogen-stimulated blastogenesis of turkey lymphocytes in the absence of serum in the culture medium. There were some differences in concentration of lymphocytes/ml of culture used in Maheswaran's and our tests. Maheswaran used 20 x 106 lymphocytes/ml of culture (6) and we used 2.1 x 100 lymphocytes/ml. The larger number of cells used by 8Millipore Corporation, Bedford, Massachusetts. 9Reeve-Angel Corporation, Clifton, New Jersey. 1OPackard-Hewlett Corporation, Downers Grove, Illinoi.
Can. J. comp. Med.
TABLE I. Effect of Addition of Fetal Calf Serum on Blastogenic Stimulation of Turkey No. 3033 Lymphocytes by Phytohemagglutinin with Polystyrene Tubes
Exp. No. 1
10% Fetal CallIf Serum in Cultures No Serum in Cultures Culture 3H-Thymidine Uptake Stimulation 3H-Thymidine bUptake Stimulation Index CPM CPMb Additionsa Index 200 4,070 2.5 PHA-P,o 13 j1 15.9 256 79 None
852 5.5 19.738 PHA-P, 13 pd 1,059 154 None 11,424 1.4 1,529 3 PHA-P, 13 ,L 936 1,116 None aO.62 x 106 lymphocytes/culture in 0.3 ml bCPM, counts per minute ,Stock solution of phytohemagglutinin (Difco, purified) in 5 ml of culture medium
18.6
2
12.2
TABLE II. Effect of Doubling Culture Volume and Contents and Varying Culture Containers on Blastogenic Stimulation of Turkey No. 3030 Lymphocytes by Phytohemagglutinin
Exp. No. 4
0.62 x 106 Lymphocytes in 0.3 ml of 1.25 x 106 Lymphocytes in 0.6 ml of Medium with 13 wl PHA-P and 10% Medium with 26 [LI PHA-P and 10% FCS FCSb Culture 3H-Thymidine Uptake Stimulation 3H-Thymidine Uptake Stimulation CPM Index Index CPMc Additionsa 13 x 100 mm Glass Test Tubes
2,014 298
PHA-P None
6.7
27.0
10,656 394
Plastic Flat Bottom Culture Plates
2.5 PHA-P 900 3,589 856 362 None RPHA-P, phytohemagglutinin (Difco, purified) In 5 ml of culture medium bFCS, fetal calf serum °CPM, counts per minute
4.2
5
TABLE III. Effects of Decantation of Culture Fluid before Turkey No. 3031 Lymphocyte Harvest and Lymphocyte Concentration on Stimulation of Blastogenesis by Phytohemagglutinin In Glass Tubes (Experiment 6)
No. Lymphocytes per 0.6 ml Culture 0.62 x 106
Culture Additionsa PHA-P,° 13 Al None
Decanted Culture Supernatant
Nondecanted Culture Supernatant
3H-Thymidine Stimulation Uptake CPMb Index 56.1 8,356 149 190.8 13,929
3H-Thymidine Stimulation Uptake
PHA-P, 26 I.l 73 None 149.3 2.5 x 106 14,187 PHA-P, 39 p1 95 None 43.0 5,038 3.75 x 106 PHA-P, 52 p.l 117 None -All cultures contained 10% fetal calf serum bCPM, counts per minute °PHA-P, phytohemagglutinin (Difco, purified) in 5 ml culture medium 1.25 x 106
Volume 42
-
April, 1978
11,930
Index 71.9
15,739
64.2
15,454
48.3
11,480
29.0
CPM
166
245
320
396
251
TABLE IV. Average Indices of PHA-Stimulated Blastogenesis of Turkey Lymphocytes Cultured in Glass Test Tubes with Morton Closures
Exp. No. 7
Conc. FCSb in Culture Medium 10%
Average for Ten Adult Turkeys
3H-Thymidine Uptake CPM° (range) 2,944 (2,1884,319) 444 (250-737) 10% 2,870 (1,371-5,217) 408 (344-648) 15% 11,079 (4,470-27,850) 526 (326-718) 15% 13,250 (2,900-20,981) 761 (507-1,648) 15% 12,598 (1,30944,830) 343 (224-353) 15% 10,446 (3,836-24,676) 251 (180-449) 1.25 x 106 lymphocytes in 0.6 ml
Culture Additionsa PHA-Pd None
Stimulation Index (range)e 6.6 (4.9-8.9)
PHA-P 7.0 (3.2-8.9) None PHA-P 9 21.1 (8.5-36) None PHA-P 10 17.4 (5.9-36) None PHA-P 11 36.7 (4.7-131) None PHA-P 12 41.6 (12.1-78) None -All cultures contained bFCS, fetal calf serum CCPM, counts per minute dPHA-P, phytohemagglutinin (Difco, purified) in 5 ml culture medium, 26 ul was added to each culture eThis figure is the average of all individual SI determinations
8
Maheswaran (6) may not have required serum as suggested by Kirchner and Oppenheim (5). Doubling the total culture volumes (including lymphocytes and PHA-P) from 0.3 to 0.6 ml also increased blastogenic stimulation (Table: II). Decanting and discarding culture supernatant fluids just before harvest of incubated lymphocytes also increased SI's (Table III). Removal of the supernatant fluids without disturbing the lymphocytes settled on the bottom of the culture tube eliminated 3H-thymidine that was unincorporated in lymphocyte DNA. This lowered detectable CPM's in control cultures and thereby ultimately raised the computed SI. Incorporating all of the above modifications, an average SI of 29.2 was obtained in cultures containing PHA-P and 1.25 x 106 lymphocytes in 0.6 ml of medium with 15% FCS in glass tubes with Morton closures (Table IV). This protocol was used to monitor lymphocyte blastogenesis stimulated by antigens of Chlamydita psittaci in turkeys vaccinated with chlamydial bacterins (L. A. Page and J. Tessler, Abstracts of Annual Meeting of American Society for Microbiology, Atlantic City, New Jersey 1976, Abst. E-135 and L. A. Page, Am. J. Vet. Res. 39: 473-480. 1978).
252
ACKNOWLEDGMENTS We thank Drs. C. C. Muscoplat and D. W. Johnson of the University of Minnesota for valuable advice and loan of cell harvesting equipment used throughout this experimentation and Mr. Joseph Wells for his technical assistance.
REFERENCES 1. ADLER, W. H., T. TAKIGUCHI, B. MARSH and R. T. SMITH. Cellular recognition by mouse lymphocytes in vitro. I. Definition of a new technique and results of stimulation by phytohemagglutinin and specific antigens. J. exp. Med. 131: 1049-1078. 1970. 2. ALHAJII, I., D. W. JOHNSON, C. C. MUSCOPLAT and C. 0. THOEN. Diagnosis of Mycobacterium bovis and MYcobacterium paratuberculosis infections in cattle by in vitro lymphocyte immunostimulation. Am. J. vet. Res. 35: 725-727. 1974. 3. BOYUM, A. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. clin. lab. Invest. 21 (suppl. 97): 77-89. 1968. 4. FERNALD, G. W. and R. S. METZGAR. In vitro studies of the response of hamster lymphocytes to phytohemagglutinin and antigenic stimulation. J. Immun. 107: 456-463. 1971. 5. KIRCHNER, H. and J. J. OPPENHEIM. Stimulation of chicken lymphocytes in serum-free medium. Cell. Immunol. 3: 695-699. 1972. 6. MAHESWARAN, S. K., E. S. THIES and C. GREIMANN. A micromethod for evaluating turkey lymphocyte responses to phytomitogens. Am. J. vet. Res. 36: 1397-1400. 1975. 7. MORTON, H. E. Stainless steel culture tube closures as a replacement for cotton plugs. Science 126: 1248. 1957. 8. NATT, M. P. and C. A. HERRICK. A new blood diluent for counting the erythrocytes and leucocytes of the chickens. Poult. Sci. 3: 735-738. 1952.
Can. J. comp. Med.
