DETERMINATION OF INTRACELLULAR CYTOKINES BY FLOW-CYTOMETRY FOLLOWING WHOLE-BLOOD CULTURE

Various methods have been reported for measuring intracellular cytokin es in peripheral blood mononuclear cells isolated by density-gradient centrifugation. In this report, we describe a whole-blood method for t he determination of intracellular cytokines (IFN-gamma, TNF-alpha and IL-2) that uses small-volume (500 mu l) blood samples. Directly conjug ated anti-cytokine antibodies and commercial cell membrane fixation an d permeabilisation reagents were used. Blood was cultured in a 1:3 dil ution with a combination of PMA and ionomycin to reveal the cytokine s ynthetic potential of each cell, together with monensin to increase th e sensitivity by retaining cytokines within the cell to detectable lev els. The optimum concentrations of PMA (10 ng/ml (16.2 nmol/l)), ionom ycin (2 mu mol/l) and monensin (3 mu mol/l) were determined. Kinetic s tudies showed maximal cytokine expression after 2 h of culture for TNF -alpha and IFN-gamma and 4 h far IL-2. Assessment of TNF-alpha and IFN -gamma production within the CD4 and CD8 lymphocytes from 10 normal vo lunteers showed that considerably more CD8+ than CD4+ cells produced I FN-gamma. This technique could be used by routine immunology laborator ies and will be of use in studies to determine whether cytokine assays are of value in the investigation of immune disorders. (C) 1997 Elsev ier Science B.V.