A quick, easy and inexpensive method for the isolation of human peripheral blood monocytes.
A commercial monocyte isolation technique based on the OptiPrep density-gradient medium was up-scaled with respect to sample and reagent volumes. The results of 7 isolations are reported in which the average purity ranged from 87.9 to 96.4%. In all but the initial isolation, monocytes were defined as CD15+ dim CD4+ dim as assessed by flow cytometric analysis; in the first isolation, monocytes were defined by the traditional CD14+ CD4+ dim combination. The mean yield (the number of isolated monocytes relative to the number present in the buffy coat) of all isolations was 26.1%, with the individual yields ranging from 10.8 to 41.4%. The mean number of isolated monocytes per experiment was 3.6 x 10(6) monocytes for those isolations performed using 14 ml of buffy coat/OptiPrep mixture (n = 4). The isolated cells were viable (> 95%) and were not activated, according to HLA-DR expression. This technique is a convenient, tast (less than 2 h), relatively simple, and inexpensive alternative to traditional monocyte isolation techniques. The up-scaled version of this method also results in significantly higher numbers of monocytes per isolation than some traditional techniques. Furthermore, this is the first literature report of the use of the OptiPrep density-gradient medium for the isolation of monocytes.
Graziani-Bowering
G M
GM
Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ont., Canada.
Graham
J M
JM
Filion
L G
LG
D015704:CD4 Antigens / Q000276:immunology; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002499:Centrifugation, Density Gradient / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D006684:HLA-DR Antigens / Q000276:immunology; D006801:Humans; D016256:Lewis X Antigen / Q000276:immunology; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D009000:Monocytes / Q000166:cytology* / Q000276:immunology*
D015704:CD4 Antigens; D006684:HLA-DR Antigens; D016256:Lewis X Antigen; D018950:Lipopolysaccharide Receptors