A new technique using an aggregating antibody against glycophorin-A for purging Ficoll-Paque-separated leucocytes of contaminating erythroid lineage cells.
Ficoll-separated leucocytes used for phenotyping may be contaminated with erythroblasts or erythrocytes, which can cause problems in cytofluorography, especially if erythroid lineage cells outnumber leucocytes in the preparation. Different means have been described for removing erythroid cells from leucocyte preparations, such as BD-FACS lysing solution, hypotonic shock and ammonium chloride, but none of these is entirely satisfactory. In my hands all these techniques damaged the leucocytes to a greater or lesser extent and did not adequately eliminate the erythroblasts, sometimes not even the reticulocytes and erythrocytes. To obtain pure leucocyte suspensions, I developed a new technique based on an aggregating antibody (alpha-Gly-A, 733/8F7, from BioCarb, Sweden) directed against glycophorin-A which is found on the cell surface of erythroid lineage cells. Agglutinated erythroid cells could be removed by filtration or low-speed centrifugation. With the new technique there was no loss or damage of the leucocytes.
Heldrup
J
J
Department of Paediatrics, University Hospital, University of Lund, Sweden.
D000328:Adult; D000906:Antibodies / Q000276:immunology*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D017252:CD3 Complex; D015704:CD4 Antigens / Q000032:analysis; D002469:Cell Separation / Q000379:methods*; D004900:Erythroblasts / Q000276:immunology*; D004912:Erythrocytes / Q000276:immunology*; D006021:Glycophorins / Q000276:immunology*; D006801:Humans; D007962:Leukocytes / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000032:analysis; D012795:Sialoglycoproteins / Q000276:immunology*
D000906:Antibodies; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D015704:CD4 Antigens; D006021:Glycophorins; D011948:Receptors, Antigen, T-Cell; D012795:Sialoglycoproteins