Increased levels of apoptosis of leukocyte subsets in cultured PBMCs compared to whole blood as shown by annexin V binding: Relevance to cytokine production

Most of the investigatory studies of cytokine production by cells have been performed on purified cells or cell. lines by measuring the secreted cytok ine levels in the bulk culture supernatant. However, results of cytokine pr oduction from isolated peripheral blood mononuclear cells, (PBMCs) cultivat ed in synthetic media, hare been reported to be inaccurate and of low repro ducibility. Isolation procedures have been shown to be toxic to certain cel ls. We hypothesised that purified cell culture techniques may result in inc reased levels of apoptosis of cells compared with whole blood culture techn iques. To compare the effects on cell viability between PBMCs and whole blo od techniques, an Annexin V binding assay was utilised, The effect of diffe rent cell concentration and serum/plasma concentrations on apoptosis levels in the Various leukocyte subsets in PBMC and whole blood cultures followin g stimulation was investigated. There mere significantly increased levels o f apoptosis of cells in PBMC compared to whole culture at similar plasma co ncentrations, suggesting that cell viability was plasma concentration-depen dent. There were significantly increased levels of apoptosis in PBMC cultur es at the same cell concentration to whole blood techniques, suggesting tha t interaction between all cellular elements (as in whole blood techniques) is important in maintaining cell viability. These results suggest that whol e blood culture techniques provide the best conditions for study of leukocy te cytokine production. If PBMC culture is performed, similar plasma and ce ll concentration to whole blood will best preserve cell viability. (C) 2000 Academic Press.