Eur. J. Immunol. 1990. 20: 201-206 Steinar Funderud+, Bjern Erikstein+, Hans Christian i(sheim+, Kjell Nustado, Trond StokkeA, Heidi Kiil BlomhoW, Harald Hoke+ and Erlend B. Smeland+ Laboratory for Immunology+, Central Laboratoryo and Department of Biophysicsa, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo
Immunomagnetic separationhsolationof human B cells
201
Functional properties of CD19+ B lymphocytes positively selected from buffy coats by immunomagnetic separation* Here we report that human B lymphocytes can be positively selected directly from buffy coats applying the anti-CD19 antibody AB1 coupled to magnetic beads. This isolation protocol is highly efficient and the isolated cell population is of very high purity and viability. As judged by cell cycle analysis and various parameters for cell activation, the cells are still in a resting state after isolation. Furthermore, different functional assays have shown that the isolation procedure does not interfere with either activation or proliferatioddifferentiation of CD19 selected cells as compared to negatively isolated cells. As a consequence of cross-linking during the isolation process, the CD19 antigen is temporarily down-regulated as measured by AB1 binding. Despite this decreased expression, monoclonal antibodies to the CD19 antigen nevertheless inhibited anti-p plus B cell growth factor induced B cell activation as reported also for negatively isolated cells. Taken together, the presented data strongly suggest that B cells isolated through the CD19 antigen can be used in critical functional assays.
1 Introduction Cells of the immune system cooperate through cell to cell interaction and through secretion of different growth and differentiation factors. To study which factors or cell interactions are essential for a certain cell entity to grow and/or differentiate, pure cell populations are required. To meet this requirement, cell separation techniques based on the molecular composition of cells have been employed, using antibodies, lectins or haptens which react directly with the desired cell population [l]. As a result of the introduction of the mAb technology in 1975 [2] and thereby antibodies defining specific lymphocyte subsets, a new generation of separation protocols have been put forward, like cell sorting by FCM [3], panning [4], a combination of panning and C-mediated lysis [5] or rosetting of cells with RBC [6]. However, after the introduction of the monosized magnetic particles by Ugelstad and co-workers [7] a new rosetting technology was developed. The surface of the particles possesses important features so that proteins can easily be adsorbed or chemically coupled, and enforce negligible nonspecific binding to cells.The versatility of this particle has been demonstrated in both positive and negative selection of T cells or T cell subsets [8-lo], in positive selection of B cells [lo] and in selection of antigen-specific B cells [111. Here we report on a simple and highly efficient method for isolation of resting B lymphocytes from buffy coats, where an anti-CD19 mAb (AB1, IgM) directly adsorbed t o
[I 79571
* This work was supported by the Norwegian Cancer Society. Correspondence: Steinar Funderud, Laboratory for Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo 3, Norway Abbreviation: BCGF: B cell growth factor 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
Dynabeads M-450 is applied. It is demonstrated that such cells are still in Go phase at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulate the vast majority of the cells to leave the Go phase, and progress through S phase in presence of growth factors.
2 Materials and methods 2.1 mAb The AB1 mAb applied in this study is an IgM antibody reactive with the CD19 antigen. Purified mAb was obtained by PEG precipitation and gel filtration of ascites fluid. HH1 is a B cell-specific mAb recognized the CD37 antigen [5]. mAb against CD4, CD8, CD4la and 1D5 (monocyte specific [12]) were kind gifts of Gustav Gaudernack, Institute of Transplantation Immunology, Oslo, Norway. OKTl1 (CD2) and OKT3 (CD3) were purchased from Ortho Pharmaceutical Corp. (Raritan, NJ). 1F5 (CD20) was kindly provided by E. Clark, Seattle, WA. NKH-1 was purchased from Coulter Immunology (Hialeah, FL). The pchain-specific mAb AF6 was a gift from R . Jeffries, Birmingham, GB, 1C10, specific for glycophorin A, was generated in our laboratory. 2.2 Isolation of B lymphocytes For positive selection of B lymphocytes, platelet-depleted buffy coats were obtained from the Blood Bank, UllevAl Hospital, Oslo, Norway. Buffy coat (50 ml) resulting from approximately 450 ml blood was transferred to a 75-cm2cell culture flask, and 0.01 M EDTA in 25 ml RPMI 1640 was added. mAb ABl-coated beads (Dynabeads M-450, Prod. no. 11002, Dynal, Oslo, Norway) were added to the cell suspension in a target-to-bead ratio of 1 : 10. The mixture was incubated on a Rock-N-Roller (Labenco, Cenco, Breda, The Netherlands) platform at 4°C for 30 min. The culture flask was than placed on a soft iron plate with ten samarium cobalt magnets (30 x 10 x 5 mm, Dynal) distri0014-2980/90/0101-0201$02.50/0
202
Eur. J. Immunol. 1990. 20: 201-206
S. Funderud, B. Erikstein, H. C. Asheim et al.
buted in such a pattern that the magnet bed covered one side wall of the flask. Rosetted cells were allowed to settle for 5 min before the liquid was drained. The cell rosettes were thereafter suspended in 10 ml of regular RE'MI 1640 containing 1% FBS and transferred to a 10-ml test tube for further washing steps. Between each washing step the rosettes were attracted to the test tube wall by a 2-min period on the magnet. After six consecutive washes in RPMI 1640 medium containing 1% FBS, 20 ml RPMI 1640 with 1% FBS was added to the cell rosettes and they were incubated for 16-20 h at 37 "C in a C02 incubator. During this incubation the AB1 beads detach from the cell membrane and the purified B lymphocytes are harvested by attracting the beads to the magnet while the cells stay in solution. For negative selection of B lymphocytes, mononuclear cells were separated from peripheral blood by gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). In order to remove all non-B cells the mononculear cell suspension was treated with Dynabeads M-450 (Prod. no. 11104) coated with mAb to CD2, CD4, CD8, 1D5, CD4la and 1C10, respectively. The mononculear cell suspension received one round of treatment with CD2, CD4 and CD8 beads on a Rock-N-Roller for 30 min at 4 "C, followed by removal of rosetted cells as described above. In a consecutive round of cell depletion, beads coated with antibodies against monocytes, erythrocytes and platelets were also included. The resulting B cell preparations were regularly > 98% pure.
2.6 Cell cycle analysis
For determination of cell cycle distribution, cells and nuclei were fixed, stained and measured as described by Stokke and co-workers [ 141. Briefly, cells or nuclei were fixed in 3% freshly prepared paraformaldehyde for at least 1 week at 0°C. Fixed cells and nuclei were washed once in 0.15 M NaCl, 0.5 mM MgC12, 10 mM Tris-HC1 (pH 7.4), 0.1% Triton X-100 and resuspended in the same buffer with 2 pg/ml Hoechst 33258 (Riedel de Haen, Seelze, FRG). This staining is stoichiometric and measures DNA content. The Hoechst 33258 fluorescence was measured by FCM upon UV excitation. Thereafter, 7-amino-actinomycin D (Calbiochem) was added to a final concentration of 25 yg/ml. Fluorescence was measured after excitation at 578 nm. To distinguish cells from first and second cell cycle, B cells were initially stimulated with anti-p plus BCGF at concentrations as given above. At 24 h BrdUrd (Sigma, St. Louis, MO), 20 pg/ml, was added to the cell culture. After 72 h of culture the cells were fixed in 25% ethanol in PBS, stored at -20°C and stained with 2pg/ml Hoechst33258 and 10 pg/ml ethidium bromide.The samples were analyzed by FCM upon UV excitation. The described staining procedure makes it possible to distinguish cells from first and second cell cycle [15].
3 Results 3.1 Separation of B lymphocytes
2.3 Staining for cell surface markers Isolated cells were analyzed for purity and expression of surface markers by indirect immunofluorescence as described elsewhere [5]. Stained cells were analyzed in a laboratory-built flow cytometer as described earlier. The data calculations are based on measurements of 30000 cells. 2.4 Cell volume determinations
Isolated cells were analyzed for initial volume distribution as well as volume increase upon stimulation by means of a modified Coulter Counter as described elsewhere [13]. The volume distribution data were based on counting of 20000 cells.
The success of the described isolation protocol is due to two main features of the CD19 antigen: expression on all B cells in peripheral blood and antigen turnover. It has previously been documented that AB1-coated particles are specifically and efficiently bound to B cell lines and normal B cells [16]. Here we extend the applicability of AB1 beads to isolation of B cells directly from buffy coats.The number of particles required for optimal rosetting of cells was titrated to a particle to target cell ratio of approximately 5 : 1. As a routine in the labortory a particle to cell ratio of 10 : 1was used. A similar ratio was also found to be optimal for Table 1. Purity of AB1-selected B cellsa) Exp. no.
Antigen (mAb)
2.5 Activation and proliferation of cells Cells were triggered to enter the cell cycle alone or in combination using the following reagents either: 75 pg/ml anti-p chain F(ab')* fragments prepared by digestion of rabbit antibodies to human Ig p chains (Dako, Copenhagen, Denmark), anti-CD20 (1F5) mAb at 2 pglml, PMA (Consolidated Midland Corporation, Brewster, NY) at a concentration of M, ionomycin (Calbiochem, La Jolla, CA), 0.5 pg/ml and partially purified low molecular weight B cell growth factor (LMW BCGF; Cellular Products, Buffalo, NY) as growth factor. The cells were grown in RPMI 1640 with 1% FBS.
CD37 (HH1) CD2 (OKTI1) CD3 (OKT3) CD56 (NKH-1)
(1DS) Second layer
-
1
>99 0.0 0.1 0.1 0.1
neg.
2 >99 0.1 0.05 0.05 0.0 neg.
3 >99 0.05 0.1 0.0 0.1 neg.
4
>99 0.1 0.05 0.1 0.1 neg.
a) The purity of AB1-selected human B cells was determined by indirect immunofluorescence microscopy. AB1-isolated cells from four different donors were pooled and incubated with mAb specific for B cells (HHl), Tcells (OKTll,OKT3), NK cells (NKH-1) and monocytes (1D.5) followed by incubation with FITC-conjugated goat anti-mouse antibody. The data are based on counting of at least lo00 cells.
Eur. J. Immunol. 1990. 20: 201-206 SECOND LAYER
Immunomagnetic separation/isolation of human B cells
I
OKT3
203
Table 2. DNA synthesis in AB1-selected B cellsa)
Stimulus
CPm
Medium Anti-p BCGF Anti-p + BCGF 1F5 1F5 + BCGF AF6 AF6 BCGF Ionomycin PMA Ionomycin PMA
1992 (1098) 8728 (5633) 6100 (2052) 32 538 (12 322) 5053 (1355) 7 145 (3803) 3865 (3435) 5457 (3667) 367 (82) 10373 (3622) 29114 (9171)
+
+
a) Isolated cells were stimulated as indicated in Sect. 2.5. DNA synthesis was measured by incorporation of [“HIdThd during the last 16 h of a 72-h culture. The data represent mean cpm (SD) from three different experiments.
1
10
xx)
loo01
lo
FlTC FLUORESCENCE (LOG SCALE) Figure 1. FCM analysis of CD19-selected B cells. Isolated cells were stained by indirect technique with the mAb 1D5 (monocytes), OKTl1 (anti-CD2), OKT3 (anti-CD3), NKH-1 (anti-CD56) and HH1 (anti-CD37). As second layer FITC-conjugated goat antimouse Ig was used.The histograms represent fluorescence intensity from 30000 cells.
cellular volume distribution as expected from resting cells. Stimulation of such cells for 24 h with anti-y plus BCGF promotes a clear shift in cellular volume which is even more pronounced after 48 h of stimulation.To further investigate the functional capacity, the B cells were triggered to enter the cell cycle by different agents as depicted in Table 2. The results demonstrate that neither anti-y chain antibodies nor BCGF alone provoke notable DNA synthesis compared to the synergistic effect of combining these two reagents. A similar synergy was found when other strong mitogenic combinations like PMA and ionomycin was used. Weak mitogenic combinations like 1FYBCGF and AF6/BCGF gave minor [3H]dThd uptake only. Although [3H]dThd data strongly indicated that cells selected through the CD19 receptor readily enter cell cycle when properly stimulated,
separation of B cells from mononuclear cell suspensions. The isolation protocol has also been applied with success to the separation of B cells from spleen and tonsils.
3.2 Cell yield and purity The described isolation method will typically yield 20 x 106-70 x lo6 B lymphocytes from a buffy coat made from 450 ml blood. A similar yield is obtained if mononuclear cells are isolated by Ficoll-Isopaque gradient centrifugation before AB1 bead separation. The isolated B cell population are consistently very pure and consist of 2 99% B lymphocytes as revealed by FCM analysis (Fig. 1) and counting of CD37+ cells in a fluorescence microscope. The purity of the isolated cells is also demonstrated by the low number of monocytes (
Immunomagnetic separationhsolationof human B cells
201
Functional properties of CD19+ B lymphocytes positively selected from buffy coats by immunomagnetic separation* Here we report that human B lymphocytes can be positively selected directly from buffy coats applying the anti-CD19 antibody AB1 coupled to magnetic beads. This isolation protocol is highly efficient and the isolated cell population is of very high purity and viability. As judged by cell cycle analysis and various parameters for cell activation, the cells are still in a resting state after isolation. Furthermore, different functional assays have shown that the isolation procedure does not interfere with either activation or proliferatioddifferentiation of CD19 selected cells as compared to negatively isolated cells. As a consequence of cross-linking during the isolation process, the CD19 antigen is temporarily down-regulated as measured by AB1 binding. Despite this decreased expression, monoclonal antibodies to the CD19 antigen nevertheless inhibited anti-p plus B cell growth factor induced B cell activation as reported also for negatively isolated cells. Taken together, the presented data strongly suggest that B cells isolated through the CD19 antigen can be used in critical functional assays.
1 Introduction Cells of the immune system cooperate through cell to cell interaction and through secretion of different growth and differentiation factors. To study which factors or cell interactions are essential for a certain cell entity to grow and/or differentiate, pure cell populations are required. To meet this requirement, cell separation techniques based on the molecular composition of cells have been employed, using antibodies, lectins or haptens which react directly with the desired cell population [l]. As a result of the introduction of the mAb technology in 1975 [2] and thereby antibodies defining specific lymphocyte subsets, a new generation of separation protocols have been put forward, like cell sorting by FCM [3], panning [4], a combination of panning and C-mediated lysis [5] or rosetting of cells with RBC [6]. However, after the introduction of the monosized magnetic particles by Ugelstad and co-workers [7] a new rosetting technology was developed. The surface of the particles possesses important features so that proteins can easily be adsorbed or chemically coupled, and enforce negligible nonspecific binding to cells.The versatility of this particle has been demonstrated in both positive and negative selection of T cells or T cell subsets [8-lo], in positive selection of B cells [lo] and in selection of antigen-specific B cells [111. Here we report on a simple and highly efficient method for isolation of resting B lymphocytes from buffy coats, where an anti-CD19 mAb (AB1, IgM) directly adsorbed t o
[I 79571
* This work was supported by the Norwegian Cancer Society. Correspondence: Steinar Funderud, Laboratory for Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo 3, Norway Abbreviation: BCGF: B cell growth factor 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
Dynabeads M-450 is applied. It is demonstrated that such cells are still in Go phase at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulate the vast majority of the cells to leave the Go phase, and progress through S phase in presence of growth factors.
2 Materials and methods 2.1 mAb The AB1 mAb applied in this study is an IgM antibody reactive with the CD19 antigen. Purified mAb was obtained by PEG precipitation and gel filtration of ascites fluid. HH1 is a B cell-specific mAb recognized the CD37 antigen [5]. mAb against CD4, CD8, CD4la and 1D5 (monocyte specific [12]) were kind gifts of Gustav Gaudernack, Institute of Transplantation Immunology, Oslo, Norway. OKTl1 (CD2) and OKT3 (CD3) were purchased from Ortho Pharmaceutical Corp. (Raritan, NJ). 1F5 (CD20) was kindly provided by E. Clark, Seattle, WA. NKH-1 was purchased from Coulter Immunology (Hialeah, FL). The pchain-specific mAb AF6 was a gift from R . Jeffries, Birmingham, GB, 1C10, specific for glycophorin A, was generated in our laboratory. 2.2 Isolation of B lymphocytes For positive selection of B lymphocytes, platelet-depleted buffy coats were obtained from the Blood Bank, UllevAl Hospital, Oslo, Norway. Buffy coat (50 ml) resulting from approximately 450 ml blood was transferred to a 75-cm2cell culture flask, and 0.01 M EDTA in 25 ml RPMI 1640 was added. mAb ABl-coated beads (Dynabeads M-450, Prod. no. 11002, Dynal, Oslo, Norway) were added to the cell suspension in a target-to-bead ratio of 1 : 10. The mixture was incubated on a Rock-N-Roller (Labenco, Cenco, Breda, The Netherlands) platform at 4°C for 30 min. The culture flask was than placed on a soft iron plate with ten samarium cobalt magnets (30 x 10 x 5 mm, Dynal) distri0014-2980/90/0101-0201$02.50/0
202
Eur. J. Immunol. 1990. 20: 201-206
S. Funderud, B. Erikstein, H. C. Asheim et al.
buted in such a pattern that the magnet bed covered one side wall of the flask. Rosetted cells were allowed to settle for 5 min before the liquid was drained. The cell rosettes were thereafter suspended in 10 ml of regular RE'MI 1640 containing 1% FBS and transferred to a 10-ml test tube for further washing steps. Between each washing step the rosettes were attracted to the test tube wall by a 2-min period on the magnet. After six consecutive washes in RPMI 1640 medium containing 1% FBS, 20 ml RPMI 1640 with 1% FBS was added to the cell rosettes and they were incubated for 16-20 h at 37 "C in a C02 incubator. During this incubation the AB1 beads detach from the cell membrane and the purified B lymphocytes are harvested by attracting the beads to the magnet while the cells stay in solution. For negative selection of B lymphocytes, mononuclear cells were separated from peripheral blood by gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). In order to remove all non-B cells the mononculear cell suspension was treated with Dynabeads M-450 (Prod. no. 11104) coated with mAb to CD2, CD4, CD8, 1D5, CD4la and 1C10, respectively. The mononculear cell suspension received one round of treatment with CD2, CD4 and CD8 beads on a Rock-N-Roller for 30 min at 4 "C, followed by removal of rosetted cells as described above. In a consecutive round of cell depletion, beads coated with antibodies against monocytes, erythrocytes and platelets were also included. The resulting B cell preparations were regularly > 98% pure.
2.6 Cell cycle analysis
For determination of cell cycle distribution, cells and nuclei were fixed, stained and measured as described by Stokke and co-workers [ 141. Briefly, cells or nuclei were fixed in 3% freshly prepared paraformaldehyde for at least 1 week at 0°C. Fixed cells and nuclei were washed once in 0.15 M NaCl, 0.5 mM MgC12, 10 mM Tris-HC1 (pH 7.4), 0.1% Triton X-100 and resuspended in the same buffer with 2 pg/ml Hoechst 33258 (Riedel de Haen, Seelze, FRG). This staining is stoichiometric and measures DNA content. The Hoechst 33258 fluorescence was measured by FCM upon UV excitation. Thereafter, 7-amino-actinomycin D (Calbiochem) was added to a final concentration of 25 yg/ml. Fluorescence was measured after excitation at 578 nm. To distinguish cells from first and second cell cycle, B cells were initially stimulated with anti-p plus BCGF at concentrations as given above. At 24 h BrdUrd (Sigma, St. Louis, MO), 20 pg/ml, was added to the cell culture. After 72 h of culture the cells were fixed in 25% ethanol in PBS, stored at -20°C and stained with 2pg/ml Hoechst33258 and 10 pg/ml ethidium bromide.The samples were analyzed by FCM upon UV excitation. The described staining procedure makes it possible to distinguish cells from first and second cell cycle [15].
3 Results 3.1 Separation of B lymphocytes
2.3 Staining for cell surface markers Isolated cells were analyzed for purity and expression of surface markers by indirect immunofluorescence as described elsewhere [5]. Stained cells were analyzed in a laboratory-built flow cytometer as described earlier. The data calculations are based on measurements of 30000 cells. 2.4 Cell volume determinations
Isolated cells were analyzed for initial volume distribution as well as volume increase upon stimulation by means of a modified Coulter Counter as described elsewhere [13]. The volume distribution data were based on counting of 20000 cells.
The success of the described isolation protocol is due to two main features of the CD19 antigen: expression on all B cells in peripheral blood and antigen turnover. It has previously been documented that AB1-coated particles are specifically and efficiently bound to B cell lines and normal B cells [16]. Here we extend the applicability of AB1 beads to isolation of B cells directly from buffy coats.The number of particles required for optimal rosetting of cells was titrated to a particle to target cell ratio of approximately 5 : 1. As a routine in the labortory a particle to cell ratio of 10 : 1was used. A similar ratio was also found to be optimal for Table 1. Purity of AB1-selected B cellsa) Exp. no.
Antigen (mAb)
2.5 Activation and proliferation of cells Cells were triggered to enter the cell cycle alone or in combination using the following reagents either: 75 pg/ml anti-p chain F(ab')* fragments prepared by digestion of rabbit antibodies to human Ig p chains (Dako, Copenhagen, Denmark), anti-CD20 (1F5) mAb at 2 pglml, PMA (Consolidated Midland Corporation, Brewster, NY) at a concentration of M, ionomycin (Calbiochem, La Jolla, CA), 0.5 pg/ml and partially purified low molecular weight B cell growth factor (LMW BCGF; Cellular Products, Buffalo, NY) as growth factor. The cells were grown in RPMI 1640 with 1% FBS.
CD37 (HH1) CD2 (OKTI1) CD3 (OKT3) CD56 (NKH-1)
(1DS) Second layer
-
1
>99 0.0 0.1 0.1 0.1
neg.
2 >99 0.1 0.05 0.05 0.0 neg.
3 >99 0.05 0.1 0.0 0.1 neg.
4
>99 0.1 0.05 0.1 0.1 neg.
a) The purity of AB1-selected human B cells was determined by indirect immunofluorescence microscopy. AB1-isolated cells from four different donors were pooled and incubated with mAb specific for B cells (HHl), Tcells (OKTll,OKT3), NK cells (NKH-1) and monocytes (1D.5) followed by incubation with FITC-conjugated goat anti-mouse antibody. The data are based on counting of at least lo00 cells.
Eur. J. Immunol. 1990. 20: 201-206 SECOND LAYER
Immunomagnetic separation/isolation of human B cells
I
OKT3
203
Table 2. DNA synthesis in AB1-selected B cellsa)
Stimulus
CPm
Medium Anti-p BCGF Anti-p + BCGF 1F5 1F5 + BCGF AF6 AF6 BCGF Ionomycin PMA Ionomycin PMA
1992 (1098) 8728 (5633) 6100 (2052) 32 538 (12 322) 5053 (1355) 7 145 (3803) 3865 (3435) 5457 (3667) 367 (82) 10373 (3622) 29114 (9171)
+
+
a) Isolated cells were stimulated as indicated in Sect. 2.5. DNA synthesis was measured by incorporation of [“HIdThd during the last 16 h of a 72-h culture. The data represent mean cpm (SD) from three different experiments.
1
10
xx)
loo01
lo
FlTC FLUORESCENCE (LOG SCALE) Figure 1. FCM analysis of CD19-selected B cells. Isolated cells were stained by indirect technique with the mAb 1D5 (monocytes), OKTl1 (anti-CD2), OKT3 (anti-CD3), NKH-1 (anti-CD56) and HH1 (anti-CD37). As second layer FITC-conjugated goat antimouse Ig was used.The histograms represent fluorescence intensity from 30000 cells.
cellular volume distribution as expected from resting cells. Stimulation of such cells for 24 h with anti-y plus BCGF promotes a clear shift in cellular volume which is even more pronounced after 48 h of stimulation.To further investigate the functional capacity, the B cells were triggered to enter the cell cycle by different agents as depicted in Table 2. The results demonstrate that neither anti-y chain antibodies nor BCGF alone provoke notable DNA synthesis compared to the synergistic effect of combining these two reagents. A similar synergy was found when other strong mitogenic combinations like PMA and ionomycin was used. Weak mitogenic combinations like 1FYBCGF and AF6/BCGF gave minor [3H]dThd uptake only. Although [3H]dThd data strongly indicated that cells selected through the CD19 receptor readily enter cell cycle when properly stimulated,
separation of B cells from mononuclear cell suspensions. The isolation protocol has also been applied with success to the separation of B cells from spleen and tonsils.
3.2 Cell yield and purity The described isolation method will typically yield 20 x 106-70 x lo6 B lymphocytes from a buffy coat made from 450 ml blood. A similar yield is obtained if mononuclear cells are isolated by Ficoll-Isopaque gradient centrifugation before AB1 bead separation. The isolated B cell population are consistently very pure and consist of 2 99% B lymphocytes as revealed by FCM analysis (Fig. 1) and counting of CD37+ cells in a fluorescence microscope. The purity of the isolated cells is also demonstrated by the low number of monocytes (
