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A multidonor ELISPOT study of IL-1β, IL-2, IL-4, IL-6, IL-13, IFN-γ and TNF-α release by cryopreserved human peripheral blood mononuclear cells

2002; Elsevier BV; Volume: 270; Issue: 2 Linguagem: Inglês

10.1016/s0022-1759(02)00297-1

1872-7905

Tanya Bailey, Sarah Stark, Angela Grant, Christopher Hartnett, Monica Tsang, Alexander E. Kalyuzhny,

T-cell and B-cell Immunology

Utilization of cryopreserved peripheral blood mononuclear cells (PBMCs), rather than fresh ones collected from the same donor on different dates, overcomes the variability in sensitivity of these cells to activation agents. To understand the effect of cryopreservation, frozen PBMCs from eight healthy donors were studied to release TH1 or TH2 cytokines including IL-1β, IL-2, IL-4, IL-6, IL-13, TNF-α and IFN-γ using ELISPOT assay. The number of spot-forming cells (SFC) was determined using three concentrations of PBMCs (5×106, 5×105 and 5×104 cells/ml). PBMCs from all eight donors were found to retain their functional capacity to release TH1 or TH2 cytokines after freezing and thawing. When PBMCs were taken in concentrations 5×106 or 5×105 cells/ml, the density of IL-1β-, IL-2-, IL-6- and TNF-α-related spots in a well for most of the donors appeared to be overly high, making SFC quantification either difficult or impossible. To the contrary, PBMCs in concentration 5×104 produced distinct and quantifiable spots. The density of spots related to IL-4 and IL-13 release appeared to be optimal for SFC quantification when PBMCs were taken in concentration 5×106 whereas in 5×105 cells/ml the spot density was very low and absent in 5×104 cells/ml concentration group. No relationship between release levels of different cytokines was found, except IFN-γ and IL-2 cytokine indicating that cryopreserved PBMCs with a high IFN-γ response will likely have a high IL-2 response as well. Our results indicate that a release level of one cytokine may not be reliably predicted by knowing the level of the other. This implies that it is necessary to test cryopreserved PBMCs in a broad range of concentrations to determine one, which will be optimal for producing distinct and quantifiable spots.