Journal of Immunological Methods, 131 (1990) 147-149
147
Elsevier JIM05638 Short communication
One step separation of human fetal lymphocytes from nucleated red blood cells Neelima M. Bhat, Marcia M. Bieber and Nelson N.H. Teng Department of Obstetrics and Gynecology, Stanford University, California, U.S.A.
(Received17 January1990,revisedreceived10 April 1990,accepted13 April 1990)
We describe a one step discontinuous density gradient centrifugation procedure to obtain a 20-fold enrichment of human fetal lymphoid cells, reducing contamination from nucleated red blood cells to less than 1%. Key words: Nucleatedred blood cell; Fetal lymphocyte;Densitygradient centrifugation
Introduction
More recently the application of specific monoclonal antibodies to analyze the cells in human fetal spleen, lymph node, thymus, and other lymphoid organs has provided an advanced way for the study of the ontogeny of lymphocytes in human lymphohemopoietic organs (Rosenthal et al., 1983). These studies mainly focus on the identification of sequential development of cellular surface phenotypes using flow cytometry (Bofill et al., 1985; Abe, 1989). Mononuclear cells from these lymphoid organs are generally obtained by Ficoll-Hypaque separation. Such preparations from fetal organs contain large numbers of nucleated red blood cells, which separate with the mononuclear cells because of their larger size and density. This contamination with nucleated red blood cells makes the quantification of the lymphoid cells in fetal and neonatal tissues difficult. It is also a handicap, while performing in
Correspondence to: N.N.H. Teng, Departmentof Obstetrics and Gynecology,Stanford University, School of Medicine, Stanford, CA 94305, U.S.A.
vitro stimulation experiments with fetal lymphocytes, where cell to cell contact is mandatory. Dextran sedimentation, followed by Ficoll-Hypaque and lysis with ammonium chloride has been described earlier to eliminate the precursors of red blood cells from neonatal blood samples (Landesberg et al., 1988). In our experience, nucleated red blood cells are resistant to lysis by ammonium chloride, and lead to inconsistent results from experiment to experiment. We have adapted the one step discontinuous density gradient centrifugation procedure described initially by English et al. to obtain a 30-fold enrichment of human fetal lymphocytes and other progenitor cells, reducing contamination from nucleated red blood cells to less than 1% (English and Anderson, 1974).
Materials and methods Tissue preparation
Fetal liver tissue samples were obtained from medically approved terminations, performed by dilation and extraction or suction curettage. Fetal age as determined by measurements of foot length ranged from 16 to 22 weeks of gestation (Trolle, 1947). Individual hepatic tissues were minced in
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
148
Mononuclearcells ~1
I ql
Nucleated RBcs
~ I - - - Mononuclearcells
I
1077-1b
MatureRBCs ~
I F:::l.---moc,eotod
~
A
F B
Fig. 1. a: single density gradient centrifugation with Histopaque-1077, b: discontinuous density gradient centrifugation with Histopaque-1077 and Histopaque-1119. medium (Hanks' balanced salt solution with 1% FCS and 0.2% DNase) and passed through sterile nylon membranes to obtain a single cell suspension.
Density gradient centrifugation Single gradient centrifugation.
9 ml of cell suspension was carefully layered onto 4 ml of Histopaque-1077 (Sigma Diagnostics, St. Louis, MO), in 15 ml sterile polystyrene tubes (Sarstedt, F.R.G.). Discontinuous density gradient. 9 ml of cell suspension was carefully layered onto a double gradient with H i s t o p a q u e - l l l 9 (Sigma Diagnostics, St. Louis, MO) at the bottom and Histopaque-1077 (2 ml each) on top, in 15 ml sterile polystyrene tubes (Sarstedt, F.R.G.) (Fig. 1). The tubes were centrifuged at 400 × g for 30 min, cells at the interfaces were recovered, washed twice with PBS, and resuspended in Hanks' balanced salt solution with 2% FCS.
Staining procedure
Fig. 2. Hematoxylin-eosin stained cells obtained from single density gradient centrifugation (1077). The arrow shows the typical morphologyof a nucleated red blood cell.
Co., Tokyo, Japan) and stained with hematoxylin-eosin as described elsewhere (Vacca, 1985).
Results and discussion Of the random 200 cells that were counted in each fraction, from three independent separations, the average number of mononuclear cells and nucleated red blood cells present in each is tabulated in Table 1. Mononuclear cells from fetal fiver that were isolated by single density gradient separation alone, yielded approximately 15-20% nucleated red blood cells as measured by hematoxylin-eosin staining (Table I, Fig. 2). The discontinuous density gradient gave a very efficient
Cytocentrifuge cell preparations were fixed in methanol (Cytocentrifuge, Sakura Fine Technical TABLE I NUMBER OF MONONUCLEARCELLS AND NUCLEATED RED BLOOD CELLS IN EACH INTERFACE a
Mononuclear cells Nucleated red blood cells Percent nucleated red blood cells
Discontinuous gradient Top Lower interface interface
Single gradient 1077
205.6+ 4.9
47.0 4-3.0
70.0+ 1.5
173.0+8.8
35.7+3.8
1 < 0.5
78.6
17.4
a Mean+ standard deviation from three different separations.
Fig. 3. Hematoxylin-eosin stained cells obtained from the lower interface of the discontinuous gradient. The arrow shows the typical morphologyof a nucleated red blood cell.
149
Thus, this technique employed to eliminate red blood cell precursors from fetal lymphocytes gives a cleaner cell preparation and will allow quantification of neonatal lymphoid cells without artifacts.
References
Fig. 4. Hematoxylin-eosin stained cells obtained from the top interface of the discontinuous gradient. Nucleated red blood cells are absent.
separation of lymphocytes (as identified by hematoxylin-eosin staining) and other progenitor cells from the nucleated red blood cells. Less than 1% nucleated reds were found in the top layer of the gradient, whereas these cells constituted about 80% of the total cells in the bottom layer (Table I, Figs. 3 and 4). The banding at the interface of the single density gradient was generally diffuse, spreading into Histopaque-1077 (Fig. 1), whereas the discontinuous gradient allowed formation of tight bands at each interface.
Abe, J. (1989) Immunocytochemical characterization of lymphocyte development in human embryonic and fetal livers. Clin. Immunol. Immunopathol. 51, 13. Bofill, M., Janossy, G., Janossa, M., Burford, G.D., Seymour, G.J., Wernet, P. and Kelemen, E. (1985) Human B cell development, subpopulation in the human fetus. J. Immunol. 134, 1531. English, D. and Anderson, B.R. (1974) Single-step separation of red blood cells. Granulocytes and mononuclear leucocytes on discontinuous density gradient of Ficoll-Hypaque. J. Immunol. Methods 5, 249. Landesberg, R., Fallon, M. and Insel, R. (1988) Alterations in helper-inducer and suppressor-inducer T-cell subsets in human neonatal blood. Immunology 65, 323. Rosenthal, P., Rimm, I.J., Ummiel, T., Griffin, J.D., Osathanondh, R., Schlossman, S.F. and Nadler, L.M. (1983) Ontogeny of human hemopoietic cells: Analysis utilizing monoclonal antibodies. J. Immunol. 31,232. Trolle, D. (1947) Age of fetus determined from its measures. Acta Obstet. Gynecol. Scand. 27, 327. Vacca, L.L. (1985) Laboratory Manual of Histochemistry. Raven Press, New York, p. 109.
147
Elsevier JIM05638 Short communication
One step separation of human fetal lymphocytes from nucleated red blood cells Neelima M. Bhat, Marcia M. Bieber and Nelson N.H. Teng Department of Obstetrics and Gynecology, Stanford University, California, U.S.A.
(Received17 January1990,revisedreceived10 April 1990,accepted13 April 1990)
We describe a one step discontinuous density gradient centrifugation procedure to obtain a 20-fold enrichment of human fetal lymphoid cells, reducing contamination from nucleated red blood cells to less than 1%. Key words: Nucleatedred blood cell; Fetal lymphocyte;Densitygradient centrifugation
Introduction
More recently the application of specific monoclonal antibodies to analyze the cells in human fetal spleen, lymph node, thymus, and other lymphoid organs has provided an advanced way for the study of the ontogeny of lymphocytes in human lymphohemopoietic organs (Rosenthal et al., 1983). These studies mainly focus on the identification of sequential development of cellular surface phenotypes using flow cytometry (Bofill et al., 1985; Abe, 1989). Mononuclear cells from these lymphoid organs are generally obtained by Ficoll-Hypaque separation. Such preparations from fetal organs contain large numbers of nucleated red blood cells, which separate with the mononuclear cells because of their larger size and density. This contamination with nucleated red blood cells makes the quantification of the lymphoid cells in fetal and neonatal tissues difficult. It is also a handicap, while performing in
Correspondence to: N.N.H. Teng, Departmentof Obstetrics and Gynecology,Stanford University, School of Medicine, Stanford, CA 94305, U.S.A.
vitro stimulation experiments with fetal lymphocytes, where cell to cell contact is mandatory. Dextran sedimentation, followed by Ficoll-Hypaque and lysis with ammonium chloride has been described earlier to eliminate the precursors of red blood cells from neonatal blood samples (Landesberg et al., 1988). In our experience, nucleated red blood cells are resistant to lysis by ammonium chloride, and lead to inconsistent results from experiment to experiment. We have adapted the one step discontinuous density gradient centrifugation procedure described initially by English et al. to obtain a 30-fold enrichment of human fetal lymphocytes and other progenitor cells, reducing contamination from nucleated red blood cells to less than 1% (English and Anderson, 1974).
Materials and methods Tissue preparation
Fetal liver tissue samples were obtained from medically approved terminations, performed by dilation and extraction or suction curettage. Fetal age as determined by measurements of foot length ranged from 16 to 22 weeks of gestation (Trolle, 1947). Individual hepatic tissues were minced in
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
148
Mononuclearcells ~1
I ql
Nucleated RBcs
~ I - - - Mononuclearcells
I
1077-1b
MatureRBCs ~
I F:::l.---moc,eotod
~
A
F B
Fig. 1. a: single density gradient centrifugation with Histopaque-1077, b: discontinuous density gradient centrifugation with Histopaque-1077 and Histopaque-1119. medium (Hanks' balanced salt solution with 1% FCS and 0.2% DNase) and passed through sterile nylon membranes to obtain a single cell suspension.
Density gradient centrifugation Single gradient centrifugation.
9 ml of cell suspension was carefully layered onto 4 ml of Histopaque-1077 (Sigma Diagnostics, St. Louis, MO), in 15 ml sterile polystyrene tubes (Sarstedt, F.R.G.). Discontinuous density gradient. 9 ml of cell suspension was carefully layered onto a double gradient with H i s t o p a q u e - l l l 9 (Sigma Diagnostics, St. Louis, MO) at the bottom and Histopaque-1077 (2 ml each) on top, in 15 ml sterile polystyrene tubes (Sarstedt, F.R.G.) (Fig. 1). The tubes were centrifuged at 400 × g for 30 min, cells at the interfaces were recovered, washed twice with PBS, and resuspended in Hanks' balanced salt solution with 2% FCS.
Staining procedure
Fig. 2. Hematoxylin-eosin stained cells obtained from single density gradient centrifugation (1077). The arrow shows the typical morphologyof a nucleated red blood cell.
Co., Tokyo, Japan) and stained with hematoxylin-eosin as described elsewhere (Vacca, 1985).
Results and discussion Of the random 200 cells that were counted in each fraction, from three independent separations, the average number of mononuclear cells and nucleated red blood cells present in each is tabulated in Table 1. Mononuclear cells from fetal fiver that were isolated by single density gradient separation alone, yielded approximately 15-20% nucleated red blood cells as measured by hematoxylin-eosin staining (Table I, Fig. 2). The discontinuous density gradient gave a very efficient
Cytocentrifuge cell preparations were fixed in methanol (Cytocentrifuge, Sakura Fine Technical TABLE I NUMBER OF MONONUCLEARCELLS AND NUCLEATED RED BLOOD CELLS IN EACH INTERFACE a
Mononuclear cells Nucleated red blood cells Percent nucleated red blood cells
Discontinuous gradient Top Lower interface interface
Single gradient 1077
205.6+ 4.9
47.0 4-3.0
70.0+ 1.5
173.0+8.8
35.7+3.8
1 < 0.5
78.6
17.4
a Mean+ standard deviation from three different separations.
Fig. 3. Hematoxylin-eosin stained cells obtained from the lower interface of the discontinuous gradient. The arrow shows the typical morphologyof a nucleated red blood cell.
149
Thus, this technique employed to eliminate red blood cell precursors from fetal lymphocytes gives a cleaner cell preparation and will allow quantification of neonatal lymphoid cells without artifacts.
References
Fig. 4. Hematoxylin-eosin stained cells obtained from the top interface of the discontinuous gradient. Nucleated red blood cells are absent.
separation of lymphocytes (as identified by hematoxylin-eosin staining) and other progenitor cells from the nucleated red blood cells. Less than 1% nucleated reds were found in the top layer of the gradient, whereas these cells constituted about 80% of the total cells in the bottom layer (Table I, Figs. 3 and 4). The banding at the interface of the single density gradient was generally diffuse, spreading into Histopaque-1077 (Fig. 1), whereas the discontinuous gradient allowed formation of tight bands at each interface.
Abe, J. (1989) Immunocytochemical characterization of lymphocyte development in human embryonic and fetal livers. Clin. Immunol. Immunopathol. 51, 13. Bofill, M., Janossy, G., Janossa, M., Burford, G.D., Seymour, G.J., Wernet, P. and Kelemen, E. (1985) Human B cell development, subpopulation in the human fetus. J. Immunol. 134, 1531. English, D. and Anderson, B.R. (1974) Single-step separation of red blood cells. Granulocytes and mononuclear leucocytes on discontinuous density gradient of Ficoll-Hypaque. J. Immunol. Methods 5, 249. Landesberg, R., Fallon, M. and Insel, R. (1988) Alterations in helper-inducer and suppressor-inducer T-cell subsets in human neonatal blood. Immunology 65, 323. Rosenthal, P., Rimm, I.J., Ummiel, T., Griffin, J.D., Osathanondh, R., Schlossman, S.F. and Nadler, L.M. (1983) Ontogeny of human hemopoietic cells: Analysis utilizing monoclonal antibodies. J. Immunol. 31,232. Trolle, D. (1947) Age of fetus determined from its measures. Acta Obstet. Gynecol. Scand. 27, 327. Vacca, L.L. (1985) Laboratory Manual of Histochemistry. Raven Press, New York, p. 109.
