Comparison of monocyte separation methods using flow cytometric analysis.
We isolated normal, nonactivated human monocytes from peripheral blood by four different methods: (1) rosetting with sheep erythrocytes pretreated with 2-aminoethylisothiouronium bromide hydrobromide (AET) followed by monoclonal antibody (OKT3 (CD3), B1 (CD19), Leu7, Leu11 (CD16] and complement treatment; (2) adherence to gelatin/plasma-coated flasks; (3) adherence to plastic dishes; and (4) separation by the Sepracell technique. We monitored these monocyte separations by determining cell recoveries, OKT4A+ lymphocyte contamination, monocyte binding to human immunodeficiency virus (HIV), number of non-specific esterase-positive cells, and proportion of mononuclear cells reactive with a battery of monoclonal antibodies specific for monocytes. Our results indicate that of the four methods compared, adherence to gelatin/plasma-coated flasks produced the highest purity, recovery, and satisfactory binding to HIV with the fewest contaminating CD4+ T cells.
Jones
B M
BM
Division of Host Factors, Centers for Disease Control, Atlanta, GA 30333.
Nicholson
J K
JK
Holman
R C
RC
Hubbard
M
M
D000911:Antibodies, Monoclonal / Q000276:immunology; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D005780:Gelatin; D006678:HIV / Q000378:metabolism; D006801:Humans; D009000:Monocytes / Q000166:cytology*; D012397:Rosette Formation
D000911:Antibodies, Monoclonal; D015214:Antigens, Differentiation, Myelomonocytic; D005780:Gelatin