Tissue Antigens (1977) 9, 227-229
NEWS LETTER
Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author($
Isolation of B and T Lymphocytes by Nylon Fiber Columns Ch. Werner, P.T. Klouda, M.C. Corrda, P. Vassalli and M. Jeannet Transplantation Immunology Unit, Division of Immunology and Allergy, Hopital Cantonal, Geneva, Switzerland
Received for publication 25 November, accepted 13 December 1976
Techniques for separation of T lymphocytes on nylon wool columns have been described using both rodent (Julius et al. 1973) and human lymphocytes (Eisen et al. 1972, Greaves & Brown 1974). I t has been shown (Trizio & Cudkowicz 1974, Betuel et al. 1976) that the B lymphocyte population which adheres to the nylon fibers of the column can be eluted without traumatizing the cells. This enriched Blymphocyte population used in a complement dependent cytotoxicity test gives a very low background of dead cells. The purpose of this communication is to describe in detail the technique of purification of T and B lymphocytes from the same blood sample. A purified lymphocyte suspension was obtained by Ficoll-Hypaque flotation from peripheral heparinized blood. The lymphocyte suspension was washed twice and resuspended in phosphate-buffered-saline (PBS) with 10%AB serum at a final concentration of 25 - 50 x lo6 lymphocytes per ml. Nylon fibers from Fenwd Leukopack (LP-1, Fenwal Laboratories) were soaked overnight in 0.2 N HCl, rinsed with distilled water and dried. Plastic 5 ml syringes were loaded with 0.5g of treated nylon fibers.
This amount of nylon was found sufficient for separation of up to 100 x lo6 lymphocytes. The columns were rinsed with 50ml of PBS, followed by 50ml of 10%AB serum in PBS prewarmed to 37'C. Both ends of the syringe barrel columns were , sealed with Parafilm@ (American Can ' Company) and the columns were incubated for 30 min at 37OC. Following incubation, the seals were removed, the columns placed vertically, and 1-2 ml of lymphocyte suspension prepared as above was run into the column, while excess PBS escaped through the needle end. The ends of the syringe were then sealed and the column incubated for 30min at 37'C. After incubation, a 21 G needle was fixed at the lower end of the syringe, which allows an outflow of approximately 2 ml of liquid per min from the column. The columns were washed with 10ml of PBS preheated to 37OC and the effluent containing the non-adherent cells (mainly T lymphocytes) was collected in a centrifuge tube. The needle was then removed and a further 50ml of PBS was passed through the column. This washing improves the purity of both the T and the B cell populations, but results in a loss of cells from both fractions. After washing,
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WERNER ET AL.
Table 1 Purification of B and T lymphocytes b y filtration through nylon fiber columns Exper No
1 2 3 4 5 6 7 8 9 10 Average
No of cells
Non-adherent cellsb (T cells) No recovered
% with surface
x lo6
x lo6
Ig
43 50 35 35 35 24 32 40 40 45
24 22 17 8 16 11 12 18 16 22
1 2 4 4 4 4 6 3 5 ND 3.6?1
Adherent cells' (B cells)
Total cell
No recovered % with surface
x
106
3.6 2.0 4.0 4.O 6.0 3 .O 2.0 2.0 2.0 9.0
Ig
84 58 80 80 60 70 70 37 80 53 67t 15
%
65 50 60 35 63 58 44 50
45 70 54+-10
a Purified lymphocyte population after Ficoll-Hypaque flotation b The non-adherent cells are those washed out wirh PBS C The adherent cells are those collected after incubation with AB serum
about 2 ml of AB serum was slowly passed through the column until all the PBS was replaced and the nylon fibers were well soaked in the serum. Both ends of the column were then sealed and the column was incubated at room temperature for 60 min. In some experiments, autologous plasma harvested after Ficoll-Hypaque separation was used instead of AB serum. Following incubation, the column was washed with 50ml of 10% AB serum in PBS while gently teasing the nylon fibers with an applicator. The effluent containing the adherent cells (mainly B lymphocytes) was collected in centrifuge tubes. The results of separation of B and T lymphocytes from 10 lymphocyte suspensions are given in Table 1. The total cell recovery was usually around 50% of the initial number of cells filtered. In the adherent cell fraction (B lymphocytes) between 50% and 80% of the cells were bearing Ig as identified by anti-lg (immunofluorescence). Only rarely were lower proportions of these cells obtained. The contamination of the Ig-bearing cells in the non-adherent fraction (T lymphocytes) did
not exceed 6%.The yield of B lymphocytes was in general around 30% of the original B-cell count. The yield of T lymphocytes was usually around 60%. The main advantages of the column separation of B and T lymphocytes lie in the simplicity of the technique and the short time required to obtain the two cell populations. If required, the separation can be carried out under sterile conditions. The column separation technique does not require any reagent which could interfere with the cell surface and the cells are not exposed to any antigens, enzymes or antisera. The final viability of cells assessed by trypan blue exclusions was in both fractions usually around 95% and the suspensions were void of platelets and cell debris. Both, the B and T enriched lymphocyte populations can be frozen and, after thawing, used for screening in a cytotoxicity assay. References Betuel H.. Gebuhrer, L., Roy, R. & Bertrand, J . (1976) Separation siquendelie des lymphocytes T et B sur fibre de nylon. (personal communication).
LYMPHOCYTE ISOLATION Eisen, S.A., Wedner, H.J. & Parker, C.W. (1972) Isolation of pure human peripheral blood Tlymphocytes using nylon wool columns. Immunol. Commun. 1,571-577. Greaves, M.F. & Brown G. (1974)Purification of human T and B lymphocytes. J . Immunol. 112,420-423. Julius, M.H., Simpson, E. & Herzenberg, L.A. (1973) A rapid method for the isolation of functional thymus-derived murine lymph* cytes. Eur. J. Immunol. 3,645-649.
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Trizio, D. & Cudkowicz, G. (1974)Separation of T and B lymphocytes by nylon wool columns: Evaluation of efficacy by functional assays in in vivo. J . Immunol. 113, 1093-1097. Address: Ch. Werner Transplantation Immunology Unit Division of Immunology and Allergy Hbpital cantonal Geneva Switzerland
NEWS LETTER
Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author($
Isolation of B and T Lymphocytes by Nylon Fiber Columns Ch. Werner, P.T. Klouda, M.C. Corrda, P. Vassalli and M. Jeannet Transplantation Immunology Unit, Division of Immunology and Allergy, Hopital Cantonal, Geneva, Switzerland
Received for publication 25 November, accepted 13 December 1976
Techniques for separation of T lymphocytes on nylon wool columns have been described using both rodent (Julius et al. 1973) and human lymphocytes (Eisen et al. 1972, Greaves & Brown 1974). I t has been shown (Trizio & Cudkowicz 1974, Betuel et al. 1976) that the B lymphocyte population which adheres to the nylon fibers of the column can be eluted without traumatizing the cells. This enriched Blymphocyte population used in a complement dependent cytotoxicity test gives a very low background of dead cells. The purpose of this communication is to describe in detail the technique of purification of T and B lymphocytes from the same blood sample. A purified lymphocyte suspension was obtained by Ficoll-Hypaque flotation from peripheral heparinized blood. The lymphocyte suspension was washed twice and resuspended in phosphate-buffered-saline (PBS) with 10%AB serum at a final concentration of 25 - 50 x lo6 lymphocytes per ml. Nylon fibers from Fenwd Leukopack (LP-1, Fenwal Laboratories) were soaked overnight in 0.2 N HCl, rinsed with distilled water and dried. Plastic 5 ml syringes were loaded with 0.5g of treated nylon fibers.
This amount of nylon was found sufficient for separation of up to 100 x lo6 lymphocytes. The columns were rinsed with 50ml of PBS, followed by 50ml of 10%AB serum in PBS prewarmed to 37'C. Both ends of the syringe barrel columns were , sealed with Parafilm@ (American Can ' Company) and the columns were incubated for 30 min at 37OC. Following incubation, the seals were removed, the columns placed vertically, and 1-2 ml of lymphocyte suspension prepared as above was run into the column, while excess PBS escaped through the needle end. The ends of the syringe were then sealed and the column incubated for 30min at 37'C. After incubation, a 21 G needle was fixed at the lower end of the syringe, which allows an outflow of approximately 2 ml of liquid per min from the column. The columns were washed with 10ml of PBS preheated to 37OC and the effluent containing the non-adherent cells (mainly T lymphocytes) was collected in a centrifuge tube. The needle was then removed and a further 50ml of PBS was passed through the column. This washing improves the purity of both the T and the B cell populations, but results in a loss of cells from both fractions. After washing,
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WERNER ET AL.
Table 1 Purification of B and T lymphocytes b y filtration through nylon fiber columns Exper No
1 2 3 4 5 6 7 8 9 10 Average
No of cells
Non-adherent cellsb (T cells) No recovered
% with surface
x lo6
x lo6
Ig
43 50 35 35 35 24 32 40 40 45
24 22 17 8 16 11 12 18 16 22
1 2 4 4 4 4 6 3 5 ND 3.6?1
Adherent cells' (B cells)
Total cell
No recovered % with surface
x
106
3.6 2.0 4.0 4.O 6.0 3 .O 2.0 2.0 2.0 9.0
Ig
84 58 80 80 60 70 70 37 80 53 67t 15
%
65 50 60 35 63 58 44 50
45 70 54+-10
a Purified lymphocyte population after Ficoll-Hypaque flotation b The non-adherent cells are those washed out wirh PBS C The adherent cells are those collected after incubation with AB serum
about 2 ml of AB serum was slowly passed through the column until all the PBS was replaced and the nylon fibers were well soaked in the serum. Both ends of the column were then sealed and the column was incubated at room temperature for 60 min. In some experiments, autologous plasma harvested after Ficoll-Hypaque separation was used instead of AB serum. Following incubation, the column was washed with 50ml of 10% AB serum in PBS while gently teasing the nylon fibers with an applicator. The effluent containing the adherent cells (mainly B lymphocytes) was collected in centrifuge tubes. The results of separation of B and T lymphocytes from 10 lymphocyte suspensions are given in Table 1. The total cell recovery was usually around 50% of the initial number of cells filtered. In the adherent cell fraction (B lymphocytes) between 50% and 80% of the cells were bearing Ig as identified by anti-lg (immunofluorescence). Only rarely were lower proportions of these cells obtained. The contamination of the Ig-bearing cells in the non-adherent fraction (T lymphocytes) did
not exceed 6%.The yield of B lymphocytes was in general around 30% of the original B-cell count. The yield of T lymphocytes was usually around 60%. The main advantages of the column separation of B and T lymphocytes lie in the simplicity of the technique and the short time required to obtain the two cell populations. If required, the separation can be carried out under sterile conditions. The column separation technique does not require any reagent which could interfere with the cell surface and the cells are not exposed to any antigens, enzymes or antisera. The final viability of cells assessed by trypan blue exclusions was in both fractions usually around 95% and the suspensions were void of platelets and cell debris. Both, the B and T enriched lymphocyte populations can be frozen and, after thawing, used for screening in a cytotoxicity assay. References Betuel H.. Gebuhrer, L., Roy, R. & Bertrand, J . (1976) Separation siquendelie des lymphocytes T et B sur fibre de nylon. (personal communication).
LYMPHOCYTE ISOLATION Eisen, S.A., Wedner, H.J. & Parker, C.W. (1972) Isolation of pure human peripheral blood Tlymphocytes using nylon wool columns. Immunol. Commun. 1,571-577. Greaves, M.F. & Brown G. (1974)Purification of human T and B lymphocytes. J . Immunol. 112,420-423. Julius, M.H., Simpson, E. & Herzenberg, L.A. (1973) A rapid method for the isolation of functional thymus-derived murine lymph* cytes. Eur. J. Immunol. 3,645-649.
229
Trizio, D. & Cudkowicz, G. (1974)Separation of T and B lymphocytes by nylon wool columns: Evaluation of efficacy by functional assays in in vivo. J . Immunol. 113, 1093-1097. Address: Ch. Werner Transplantation Immunology Unit Division of Immunology and Allergy Hbpital cantonal Geneva Switzerland
