Peripheral blood mononuclear cells (PBMC), which contain CD8 T cells, are stimulated with a mixture of phorbol myristate acetate (PMA) and ionomycin, and with stimulatory signals derived using antibodies against the costimulatory molecules CD28/CD49d. The cells are simultaneously treated with a mixture of brefeldin A (BFA) and monensin, which blocks extracellular secretion of interferon-gamma (IFN-gamma), enabling intracellular retention and detection of the protein. PBMCs that have not been stimulated are used as a control to determine the background levels of IFN-gamma and CD107a and CD107b. The cells are analyzed on the BD FACSCanto flow cytometer and analysis involves gating (defining) of the CD8 T cells using an antihuman CD8 antibody. Specific IFN-gamma and CD107a and CD107b signals are determined within the "gated" CD8 T-cell population. Global CD8 T-cell immune competence is measured by the amount of IFN-gamma produced (CD8 T-cell functional activity) and surface expression of CD107a and CD107b (cytotoxicity assessment) relative to the unstimulated control and is interpreted on the basis of the reference range determined from healthy adult donors.(Unpublished Mayo method)
Monday through Friday
Specimens are required to be received in the laboratory on weekdays and by 4 p.m. on Friday. No weekend processing
PBMC's are stored for 7 days at -70 degrees C.