Analysis of TNF-α production by γδ T cells in melanoma patients and age-matched healthy subjects. PBMC young/adult and old melanoma patients and young/adult and old healthy subjects were stimulated for 18 h in the presence of IPP (30 μg per ml) and IL-2 (100 U per ml). The last 12 h of culture were performed in the presence of GolgiPlug, a protein transport inhibitor containing brefeldin. Single-cell analysis of TNF-α synthesis in γδ T cells from a representative subject for each group was performed following dual staining with cell surface anti- γδ T (FITC) MoAb and intracellular anti- TNF-α (PE) MoAb. Number in brackets indicate the percentages of γδ T cells synthesizing TNF-α among total γδ T lymphocytes.