title: Increased levels of apoptosis of leukocyte subsets in cultured PBMCs compared to whole blood as shown by Annexin V binding: relevance to cytokine production.
abstract: Most of the investigatory studies of cytokine production by cells have been performed on purified cells or cell lines by measuring the secreted cytokine levels in the bulk culture supernatant. However, results of cytokine production from isolated peripheral blood mononuclear cells (PBMCs) cultivated in synthetic media, have been reported to be inaccurate and of low reproducibility. Isolation procedures have been shown to be toxic to certain cells. We hypothesised that purified cell culture techniques may result in increased levels of apoptosis of cells compared with whole blood culture techniques. To compare the effects on cell viability between PBMCs and whole blood techniques, an Annexin V binding assay was utilised. The effect of different cell concentration and serum/plasma concentrations on apoptosis levels in the various leukocyte subsets in PBMC and whole blood cultures following stimulation was investigated. There were significantly increased levels of apoptosis of cells in PBMC compared to whole culture at similar plasma concentrations, suggesting that cell viability was plasma concentration-dependent. There were significantly increased levels of apoptosis in PBMC cultures at the same cell concentration to whole blood techniques, suggesting that interaction between all cellular elements (as in whole blood techniques) is important in maintaining cell viability. These results suggest that whole blood culture techniques provide the best conditions for study of leukocyte cytokine production. If PBMC culture is performed, similar plasma and cell concentration to whole blood will best preserve cell viability.
authors.affiliation: Haematology Department, Adelaide Women's and Children's Hospital, North Adelaide, South Australia, 5006, Australia.
authors.forename: G
authors.initials: G
authors.lastname: Hodge
authors.forename: S
authors.initials: S
authors.lastname: Hodge
authors.forename: P
authors.initials: P
authors.lastname: Han
mesh_terms: D000328:Adult; D017304:Annexin A5 / Q000097:blood* / Q000378:metabolism*; D017209:Apoptosis; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis* / Q000097:blood*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D007694:Killer Cells, Natural; D007962:Leukocytes / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism; D008213:Lymphocyte Activation; D009000:Monocytes / Q000378:metabolism; D010835:Phytohemagglutinins / Q000378:metabolism; D011485:Protein Binding; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: D017304:Annexin A5; D003470:Culture Media; D016207:Cytokines; D008070:Lipopolysaccharides; D010835:Phytohemagglutinins