Removal of erythroid cells from umbilical cord blood mononuclear cell preparations using magnetic beads and a monoclonal antibody against glycophorin A.
Umbilical cord blood mononuclear cells isolated by density centrifugation are contaminated by erythrocytes and nucleated erythroid precursors which may exceed 50% of the total cell population, and thus interfere with phenotypic, functional and mRNA analyses. Lysis with hypotonic ammonium chloride can overcome this problem, but interferes with lysosomal function and should be avoided when cell preparations are intended for functional studies. The aim of this study was to develop a technique for removing erythroid cells from cord blood mononuclear cell preparations that would be as effective as ammonium chloride lysis but would not affect cellular function. This paper describes a method using 10F7, a mouse monoclonal antibody against human glycophorin A, and magnetic beads coated with anti-mouse immunoglobulin. The population of cord blood mononuclear cells recovered using this technique was of high purity, good yield and viability, and the cells responded appropriately to stimulation in vitro. To maximise cost-effectiveness, purification with magnetic beads could be performed after two density separations to reduce the quantity of beads required.
Child Health Research Institute, Women's and Children's Hospital, Adelaide, South Australia, Australia.
S R
SR
Elliott
P J
PJ
Macardle
H
H
Zola
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D004912:Erythrocytes / Q000276:immunology; D005069:Evaluation Studies as Topic; D005312:Fetal Blood / Q000166:cytology*; D006021:Glycophorins / Q000276:immunology*; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology; D018189:Immunomagnetic Separation; D007231:Infant, Newborn; D007963:Leukocytes, Mononuclear; D051379:Mice; D009996:Osmotic Fragility
D000911:Antibodies, Monoclonal; D006021:Glycophorins; D007038:Hypotonic Solutions; D000643:Ammonium Chloride