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Influence of erythrocyte contamination on the optimal phytohaemagglutinin concentration in Chinese hamster lymphocyte cultures
Journal of Immunological Methods, 61 (1983) 43-48
43
Elsevier
Influence of Erythrocyte Contamination on the Optimal Phytohaemagglutinin Concentration in Chinese Hamster Lymphocyte Cultures B. De Jong, G.J.P.A. Anders, V.J.S. Idenburg and J. Zijlstra Anthropogenetisch lnstituut, Antonius Deusinglaan 4, 9713 A W Groningen, The Netherlands
(Received 23 August 1982, accepted 4 January 1983)
Lymphocyte cultures from the Chinese hamster were made with lymphocytes isolated with Ficoll-Isopaque or with Haemaccel. In some experiments the red blood cells (r.b.c.) that remained after lymphocyte isolation were lysed. Optimum phytohaemagglutinin (PHA) concentration for lymphocyte proliferation depended upon the manner in which the lymphocytes were obtained. Lower PHA concentrations were needed for optimum stimulation of lymphocytes isolated with Ficoll-Isopaque than for lymphocytes isolated with Haemaccel. Lysis of the r.b.c, remaining after isolation of lymphocytes with Haemaccel resulted in a decrease of optimum PHA concentration. The optimum PHA concentration depended strongly on r.b.c, contamination, the higher the r.b.c, contamination the higher the optimum PHA concentration. However, only part of the differences found in optimum PHA concentrations can be attributed to r.b.c contamination. Key words: phytohaemagglutinin - - lymphocyte culture conditions - - lymphocyte stimulation
Introduction Chinese h a m s t e r l y m p h o c y t e s m a y b e s t i m u l a t e d b y P H A in total b l o o d cultures ( D e Jong a n d A n d e r s , 1972) or in cultures with isolated l y m p h o c y t e s ( D e J o n g et al., 1976). L y m p h o c y t e s m a y be isolated b y different m e t h o d s (Ling a n d K a y , 1975). W e used gelatine s e d i m e n t a t i o n , b u t o t h e r s e p a r a t i o n m e t h o d s ( F i c o l l - I s o p a q u e isolation) ( T h o r s b y a n d Bratlie, 1970) a n d red b l o o d cell lysis ( C h a n d l e r 1980) are also suitable. W i t h the different l y m p h o c y t e isolation m e t h o d s we o b s e r v e d striking differences in r.b.c, c o n t a m i n a t i o n a n d o p t i m u m P H A c o n c e n t r a t i o n for l y m p h o c y t e proliferation. Because it is k n o w n (H~iyry a n d Defendi, 1970; Tiirnvik, 1970; Y a c h n i n et al., 1971) that r.b.c, m a y b o t h e n h a n c e a n d inhibit l y m p h o c y t e p r o l i f e r a t i o n we exa m i n e d the role o f r.b.c, in Chinese h a m s t e r l y m p h o c y t e proliferation. 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.
44 Materials and Methods
From each hamster about 0.8 ml of blood was taken by puncture from the retrobulbar venous plexus and collected in a heparinized culture tube (Falcon tube 2003). Usually blood samples from several animals were pooled. For gelatine sedimentation the blood was mixed with approximately 3 times the volume of Haemaccel (Behringwerke) and allowed to stand at room temperature. After about 1 h the supernatant was transfered to culture tubes, mixed with approximately one third the volume of RPMI 1640 with Hepes (Gibco) and centrifuged for 10 min at 240 × g. The cells were washed twice with RPMI 1640 with Hepes and resuspended in culture medium. The density of Haemaccel is 1.010 g/ml. Lymphocyte isolation on Ficoll-Isopaque was by centrifuging an equal mixture of blood and physiological saline on Lymphoprep (Nyegaard, Oslo) for 20 min at 1000 × g. To 100 ml Lymphoprep 2.2 g Ficoll (Pharmacia) was added giving a density of 1.083 g/ml. With Haemaccel sedimentation and Ficoll-Isopaque isolation the yield of leucocytes was approximately the same, being about 1.9 × 106 white blood cells (w.b.c.) per hamster, with an average of 90% lymphocytes. After Haemaccel sedimentation contamination with red blood cells (r.b.c.) was approximately 60 r.b.c. : 1 w.b.c, and after Ficoll-Isopaque sedimentation 0.8 r.b.c. : 1 w.b.c. In some experiments, after Haemaccel isolation of the lymphocytes, the remaining r.b.c, were lysed according to the method of Chandler (1980). After lysis r.b.c. contamination was about the same as with Ficoll-Isopaque isolation without lysis. The cells were cultured in RPMI 1640 with Hepes (Gibco) supplemented with streptomycin (100 #g/ml), penicillin (100 I U / m l ) , 20% heat inactivated foetal calf serum (Gibco) and 2-mercaptoethanol at a final concentration of 40/~M. The cells were stimulated with phytohaemagglutinin (PHA Wellcome HA 15, 10-140 /~l/ml culture) in a total culture volume of 0.1 ml in Cooke microtitre plates with V-bottomed wells (cat. no. 25 AR) for 3 days in a humidified atmosphere with 5% CO 2 in air at 37°C. The cultures were labelled with 2/~Ci [3H]thymidine (specific activity 2 C i / m m o l ) added in 0.02 ml RPMI 1640 with Hepes and harvested 16-24 h later with a multiple cell culture harvester with glass fiber filters. The filters were dried and transferred to counting vials containing 5 ml of a liquid scintillation solution. Results are expressed as mean values of 3 replicate cultures, in disintegrations per minute (dpm) + S.D. All experiments were done at least 3 times. The viability of the cells, as determined with trypan blue, was between 96-100%.
Results
In the experiments in Fig. 1 lymphocytes were isolated with Haemaccel (H lymphocytes); with Ficoll-Isopaque (F lymphocytes) and with Haemaccel followed by lysis of the r.b.c. (H + L lymphocytes). The figure shows that the PHA concentrations needed for optimum lymphocyte stimulation are different for lymphocytes obtained by different isolation procedures.
45
°P"
[
X 104
t
,o
1 lO
~l
PHA,,,). 0
2
4
6
8
10
12
14
Fig. I. Stimulation of l0 5 lymphocytes isolated with Haemaccel (*), Haemaccel followed by lysis of the r.b.c. (D) and Ficoll-Isopaque (e). The 105 lymphocytes/0.l ml culture were stimulated with 0, 2, 4, 6, 8, 10, 12 or 14 #1 PHA/0.1 ml culture. DPM X 104
20
10
~ul PHA-.]~
0
t
2
4
e
e
10
12
~4
Fig. 2. Addition of 0 r.b.c. (©), 25 r.b.c. (0), 50 r.b.c. (*) and 100 r.b.c. (n) to one w.b.c, in cultures of 0.1 ml containing 105 lymphocytes isolated with Ficoll-Isopaque. The iymphocytes were stimulated with 0-14 #1 PHA/0.1 ml culture.
46 Lowest optimal PHA concentrations were in cultures with (H + L) lymphocytes and highest PHA concentrations in H lymphocyte cultures. Optimal PHA concentrations for F lymphocytes were intermediate. It may be seen that the optimal PHA concentration for lymphocytes separated by one method may be strongly inhibitory for lymphocytes separated by another method. One of the most striking differences between H, F and H + L lymphocytes was the contamination with r.b.c. With H lymphocytes this was about 60 r.b.c, for each w.b.c, and with F and H + L lymphocytes about 1 r.b.c, to 1 w.b.c. To examine the influence of r.b.c, on optimal PHA concentration, r.b.c, were added to F and H + L lymphocytes. As seen in Figs. 2 and 3, addition of r.b.c, results in a shift of the optimal PHA concentration to higher values. Addition of more than about 100 r.b.c, to 1 w.b.c. resulted in a decrease of lymphocyte proliferation. Thus, part of the differences in optimal PHA concentration was traced to differences in r.b.c, contamination. However, the differences in optimal PHA concentrations between H + L and F lymphocytes, both with about the same r.b.c. contamination, indicate that probably the composition of the different lymphocyte
DPM X 104
10
L PHA,qb
0
1
2
4
s
8
10
12
14
Fig. 3. Addition of 0 r.b.c. (O), 25 r.b.c. (e) 50 r.b.c. (*) and 100 r.b.c. (D) to 1 w.b.c, in cultures of 0.1 ml with 105 lymphocytesisolated with Haemaccel followed by lysls of the remaining r.b.c. The lymphocytes were stimulated with 0-14/tl PHA/0.1 ml culture.
47 populations is also important. With the different isolation procedures the yield of lymphocytes is about 20-30% of the total lymphocytes in blood, and the composition of the lymphocyte populations obtained may differ. Discussion
The effect of erythrocytes in lymphocyte cultures has been described for man and different animals. T~irnvik (1970); Yachnin et al. (1971) and Johnson et al. (1972) reported enhancement of stimulation of human lymphocytes by PHA when red cells or red cell stroma were added to the lymphocyte cultures. H~iyry and Defendi (1970) described inhibition of the mouse mixed lymphocyte reaction (MLR) in suspensions that contain more than 25-50 r.b.c./w.b.c. Brooks (1975) described inhibition of mouse M L R by syngeneic r.b.c, but a pronounced enhancement of guinea pig lymphocyte responses to PHA in the presence of syngeneic r.b.c. In the Chinese hamster we observed that with increasing r.b.c, contamination increasing amounts of PHA were needed to obtain optimal lymphocyte stimulation. Sometimes the addition of low amounts of r.b.c, gave enhancement of lymphocyte proliferation whereas addition of more than 50-100 r.b.c./w.b.c, gave inhibition. Enhancement of lymphocyte proliferation by addition of r.b.c, is probably brought about by the binding of PHA to erythrocytes. As a result the PHA displays a steric configuration, and reaches a local concentration favouring lymphocyte stimulation (Johnson et al., 1972; Ling and Kay, 1975). Binding of PHA to r.b.c, is probably also the reason for the shift to higher optimal PHA concentrations with increasing r.b.c, contamination. Inhibition of lymphocyte stimulation produced by the addition of too many r.b.c. may be caused by diminished possibilities for the cell contact necessary for induction of lymphocyte proliferation (Peters, 1972, 1974). In some experiments although the r.b.c, contamination of the lymphocytes after different isolation procedures was the same, differences in optimal PHA concentration were observed. These were probably due to different constitutions of the lymphocyte populations after the different isolation procedures. The different isolation procedures yield lymphocytes of different densities. Although Schut et al. (1978) found similar dose response curves and time courses for light and heavy lymphocytes in response to PHA, they also found, as did Ulmer and Flad (1979), differences in thymidine uptake with PHA stimulation of lymphocytes of different densities. Differences in both isolation procedure and r.b.c, contamination certainly influence culture condition and kinetics of cell death (Bernheim et al., 1977) and in this way influence also PHA reactivity and the PHA dose response curve.
References
Bernheim, J.L., J. Mendelsohn, M.F. Kelleyand R. Dorian, 1977, Proc. Natl. Acad. Sci. U.S.A. 74, 2536. Brooks, G.G., 1975,J. Immunol. Methods 9, 171. Chandler, P., 1980, Immunol. Today 1, 82.
48 De Jong, B. and G.J.P.A. Anders, 1972, Experientia 28, 86. De Jong, B., G.J.P.A. Anders, I.H. Van der Meer and J. Zijlstra, 1976, J. Immunol. Methods 12, 91. Hayry, P. and V. Defendi, 1970, Clin. Exp. Immunol. 6, 345. Johnson, R.A., T.K. Smith and C.H. Kirkpatrick, 1972, Cell. Immunol. 3, 186. Ling, N.R. and J.E. Kay, 1975, Lymphocyte Stimulation (North-Holland, Amsterdam). Peters, J.H., 1972, Exp. Cell Res. 74, 179. Peters, J.H., 1974, On the Hypothesis of Cell Contact Mediated Lymphocyte Stimulation. Lymphocyte Recognition and Effect or Mechanism (Academic Press, New York) p. 13. Schut, B.J.T., H.A. Loos, M.T.L. Roos, T. Pinkster and W.P. Zeylemaker, 1979, Eur.J. lmmunol. 8, 1. T~rnvik, A., 1970, Acta Pathol. Microbiol. Scand. Sect. B 78, 733. Thorsby, E. and A. Bratlie, 1970, A Rapid Method for Preparation of Pure Lymphocyte Suspensions. Histocompatibility Testing (Munksgaard, Copenhagen) p. 655. Ulmer, A.J. and H.-D. Flad, 1979, J. Immunol. Methods 30, 1. Yachnin, S., L.W. Allen, J.M. Baron and R. Svenson, 1971, Potentiation of Lymphocyte Transformation by Membrane-Membrane Interaction. Fourth Annual Leucocyte Conference (Appleton-CenturyCrofts, New York).
43
Elsevier
Influence of Erythrocyte Contamination on the Optimal Phytohaemagglutinin Concentration in Chinese Hamster Lymphocyte Cultures B. De Jong, G.J.P.A. Anders, V.J.S. Idenburg and J. Zijlstra Anthropogenetisch lnstituut, Antonius Deusinglaan 4, 9713 A W Groningen, The Netherlands
(Received 23 August 1982, accepted 4 January 1983)
Lymphocyte cultures from the Chinese hamster were made with lymphocytes isolated with Ficoll-Isopaque or with Haemaccel. In some experiments the red blood cells (r.b.c.) that remained after lymphocyte isolation were lysed. Optimum phytohaemagglutinin (PHA) concentration for lymphocyte proliferation depended upon the manner in which the lymphocytes were obtained. Lower PHA concentrations were needed for optimum stimulation of lymphocytes isolated with Ficoll-Isopaque than for lymphocytes isolated with Haemaccel. Lysis of the r.b.c, remaining after isolation of lymphocytes with Haemaccel resulted in a decrease of optimum PHA concentration. The optimum PHA concentration depended strongly on r.b.c, contamination, the higher the r.b.c, contamination the higher the optimum PHA concentration. However, only part of the differences found in optimum PHA concentrations can be attributed to r.b.c contamination. Key words: phytohaemagglutinin - - lymphocyte culture conditions - - lymphocyte stimulation
Introduction Chinese h a m s t e r l y m p h o c y t e s m a y b e s t i m u l a t e d b y P H A in total b l o o d cultures ( D e Jong a n d A n d e r s , 1972) or in cultures with isolated l y m p h o c y t e s ( D e J o n g et al., 1976). L y m p h o c y t e s m a y be isolated b y different m e t h o d s (Ling a n d K a y , 1975). W e used gelatine s e d i m e n t a t i o n , b u t o t h e r s e p a r a t i o n m e t h o d s ( F i c o l l - I s o p a q u e isolation) ( T h o r s b y a n d Bratlie, 1970) a n d red b l o o d cell lysis ( C h a n d l e r 1980) are also suitable. W i t h the different l y m p h o c y t e isolation m e t h o d s we o b s e r v e d striking differences in r.b.c, c o n t a m i n a t i o n a n d o p t i m u m P H A c o n c e n t r a t i o n for l y m p h o c y t e proliferation. Because it is k n o w n (H~iyry a n d Defendi, 1970; Tiirnvik, 1970; Y a c h n i n et al., 1971) that r.b.c, m a y b o t h e n h a n c e a n d inhibit l y m p h o c y t e p r o l i f e r a t i o n we exa m i n e d the role o f r.b.c, in Chinese h a m s t e r l y m p h o c y t e proliferation. 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.
44 Materials and Methods
From each hamster about 0.8 ml of blood was taken by puncture from the retrobulbar venous plexus and collected in a heparinized culture tube (Falcon tube 2003). Usually blood samples from several animals were pooled. For gelatine sedimentation the blood was mixed with approximately 3 times the volume of Haemaccel (Behringwerke) and allowed to stand at room temperature. After about 1 h the supernatant was transfered to culture tubes, mixed with approximately one third the volume of RPMI 1640 with Hepes (Gibco) and centrifuged for 10 min at 240 × g. The cells were washed twice with RPMI 1640 with Hepes and resuspended in culture medium. The density of Haemaccel is 1.010 g/ml. Lymphocyte isolation on Ficoll-Isopaque was by centrifuging an equal mixture of blood and physiological saline on Lymphoprep (Nyegaard, Oslo) for 20 min at 1000 × g. To 100 ml Lymphoprep 2.2 g Ficoll (Pharmacia) was added giving a density of 1.083 g/ml. With Haemaccel sedimentation and Ficoll-Isopaque isolation the yield of leucocytes was approximately the same, being about 1.9 × 106 white blood cells (w.b.c.) per hamster, with an average of 90% lymphocytes. After Haemaccel sedimentation contamination with red blood cells (r.b.c.) was approximately 60 r.b.c. : 1 w.b.c, and after Ficoll-Isopaque sedimentation 0.8 r.b.c. : 1 w.b.c. In some experiments, after Haemaccel isolation of the lymphocytes, the remaining r.b.c, were lysed according to the method of Chandler (1980). After lysis r.b.c. contamination was about the same as with Ficoll-Isopaque isolation without lysis. The cells were cultured in RPMI 1640 with Hepes (Gibco) supplemented with streptomycin (100 #g/ml), penicillin (100 I U / m l ) , 20% heat inactivated foetal calf serum (Gibco) and 2-mercaptoethanol at a final concentration of 40/~M. The cells were stimulated with phytohaemagglutinin (PHA Wellcome HA 15, 10-140 /~l/ml culture) in a total culture volume of 0.1 ml in Cooke microtitre plates with V-bottomed wells (cat. no. 25 AR) for 3 days in a humidified atmosphere with 5% CO 2 in air at 37°C. The cultures were labelled with 2/~Ci [3H]thymidine (specific activity 2 C i / m m o l ) added in 0.02 ml RPMI 1640 with Hepes and harvested 16-24 h later with a multiple cell culture harvester with glass fiber filters. The filters were dried and transferred to counting vials containing 5 ml of a liquid scintillation solution. Results are expressed as mean values of 3 replicate cultures, in disintegrations per minute (dpm) + S.D. All experiments were done at least 3 times. The viability of the cells, as determined with trypan blue, was between 96-100%.
Results
In the experiments in Fig. 1 lymphocytes were isolated with Haemaccel (H lymphocytes); with Ficoll-Isopaque (F lymphocytes) and with Haemaccel followed by lysis of the r.b.c. (H + L lymphocytes). The figure shows that the PHA concentrations needed for optimum lymphocyte stimulation are different for lymphocytes obtained by different isolation procedures.
45
°P"
[
X 104
t
,o
1 lO
~l
PHA,,,). 0
2
4
6
8
10
12
14
Fig. I. Stimulation of l0 5 lymphocytes isolated with Haemaccel (*), Haemaccel followed by lysis of the r.b.c. (D) and Ficoll-Isopaque (e). The 105 lymphocytes/0.l ml culture were stimulated with 0, 2, 4, 6, 8, 10, 12 or 14 #1 PHA/0.1 ml culture. DPM X 104
20
10
~ul PHA-.]~
0
t
2
4
e
e
10
12
~4
Fig. 2. Addition of 0 r.b.c. (©), 25 r.b.c. (0), 50 r.b.c. (*) and 100 r.b.c. (n) to one w.b.c, in cultures of 0.1 ml containing 105 lymphocytes isolated with Ficoll-Isopaque. The iymphocytes were stimulated with 0-14 #1 PHA/0.1 ml culture.
46 Lowest optimal PHA concentrations were in cultures with (H + L) lymphocytes and highest PHA concentrations in H lymphocyte cultures. Optimal PHA concentrations for F lymphocytes were intermediate. It may be seen that the optimal PHA concentration for lymphocytes separated by one method may be strongly inhibitory for lymphocytes separated by another method. One of the most striking differences between H, F and H + L lymphocytes was the contamination with r.b.c. With H lymphocytes this was about 60 r.b.c, for each w.b.c, and with F and H + L lymphocytes about 1 r.b.c, to 1 w.b.c. To examine the influence of r.b.c, on optimal PHA concentration, r.b.c, were added to F and H + L lymphocytes. As seen in Figs. 2 and 3, addition of r.b.c, results in a shift of the optimal PHA concentration to higher values. Addition of more than about 100 r.b.c, to 1 w.b.c. resulted in a decrease of lymphocyte proliferation. Thus, part of the differences in optimal PHA concentration was traced to differences in r.b.c, contamination. However, the differences in optimal PHA concentrations between H + L and F lymphocytes, both with about the same r.b.c. contamination, indicate that probably the composition of the different lymphocyte
DPM X 104
10
L PHA,qb
0
1
2
4
s
8
10
12
14
Fig. 3. Addition of 0 r.b.c. (O), 25 r.b.c. (e) 50 r.b.c. (*) and 100 r.b.c. (D) to 1 w.b.c, in cultures of 0.1 ml with 105 lymphocytesisolated with Haemaccel followed by lysls of the remaining r.b.c. The lymphocytes were stimulated with 0-14/tl PHA/0.1 ml culture.
47 populations is also important. With the different isolation procedures the yield of lymphocytes is about 20-30% of the total lymphocytes in blood, and the composition of the lymphocyte populations obtained may differ. Discussion
The effect of erythrocytes in lymphocyte cultures has been described for man and different animals. T~irnvik (1970); Yachnin et al. (1971) and Johnson et al. (1972) reported enhancement of stimulation of human lymphocytes by PHA when red cells or red cell stroma were added to the lymphocyte cultures. H~iyry and Defendi (1970) described inhibition of the mouse mixed lymphocyte reaction (MLR) in suspensions that contain more than 25-50 r.b.c./w.b.c. Brooks (1975) described inhibition of mouse M L R by syngeneic r.b.c, but a pronounced enhancement of guinea pig lymphocyte responses to PHA in the presence of syngeneic r.b.c. In the Chinese hamster we observed that with increasing r.b.c, contamination increasing amounts of PHA were needed to obtain optimal lymphocyte stimulation. Sometimes the addition of low amounts of r.b.c, gave enhancement of lymphocyte proliferation whereas addition of more than 50-100 r.b.c./w.b.c, gave inhibition. Enhancement of lymphocyte proliferation by addition of r.b.c, is probably brought about by the binding of PHA to erythrocytes. As a result the PHA displays a steric configuration, and reaches a local concentration favouring lymphocyte stimulation (Johnson et al., 1972; Ling and Kay, 1975). Binding of PHA to r.b.c, is probably also the reason for the shift to higher optimal PHA concentrations with increasing r.b.c, contamination. Inhibition of lymphocyte stimulation produced by the addition of too many r.b.c. may be caused by diminished possibilities for the cell contact necessary for induction of lymphocyte proliferation (Peters, 1972, 1974). In some experiments although the r.b.c, contamination of the lymphocytes after different isolation procedures was the same, differences in optimal PHA concentration were observed. These were probably due to different constitutions of the lymphocyte populations after the different isolation procedures. The different isolation procedures yield lymphocytes of different densities. Although Schut et al. (1978) found similar dose response curves and time courses for light and heavy lymphocytes in response to PHA, they also found, as did Ulmer and Flad (1979), differences in thymidine uptake with PHA stimulation of lymphocytes of different densities. Differences in both isolation procedure and r.b.c, contamination certainly influence culture condition and kinetics of cell death (Bernheim et al., 1977) and in this way influence also PHA reactivity and the PHA dose response curve.
References
Bernheim, J.L., J. Mendelsohn, M.F. Kelleyand R. Dorian, 1977, Proc. Natl. Acad. Sci. U.S.A. 74, 2536. Brooks, G.G., 1975,J. Immunol. Methods 9, 171. Chandler, P., 1980, Immunol. Today 1, 82.
48 De Jong, B. and G.J.P.A. Anders, 1972, Experientia 28, 86. De Jong, B., G.J.P.A. Anders, I.H. Van der Meer and J. Zijlstra, 1976, J. Immunol. Methods 12, 91. Hayry, P. and V. Defendi, 1970, Clin. Exp. Immunol. 6, 345. Johnson, R.A., T.K. Smith and C.H. Kirkpatrick, 1972, Cell. Immunol. 3, 186. Ling, N.R. and J.E. Kay, 1975, Lymphocyte Stimulation (North-Holland, Amsterdam). Peters, J.H., 1972, Exp. Cell Res. 74, 179. Peters, J.H., 1974, On the Hypothesis of Cell Contact Mediated Lymphocyte Stimulation. Lymphocyte Recognition and Effect or Mechanism (Academic Press, New York) p. 13. Schut, B.J.T., H.A. Loos, M.T.L. Roos, T. Pinkster and W.P. Zeylemaker, 1979, Eur.J. lmmunol. 8, 1. T~rnvik, A., 1970, Acta Pathol. Microbiol. Scand. Sect. B 78, 733. Thorsby, E. and A. Bratlie, 1970, A Rapid Method for Preparation of Pure Lymphocyte Suspensions. Histocompatibility Testing (Munksgaard, Copenhagen) p. 655. Ulmer, A.J. and H.-D. Flad, 1979, J. Immunol. Methods 30, 1. Yachnin, S., L.W. Allen, J.M. Baron and R. Svenson, 1971, Potentiation of Lymphocyte Transformation by Membrane-Membrane Interaction. Fourth Annual Leucocyte Conference (Appleton-CenturyCrofts, New York).