Cytoplasmic expression of IFN-γ and TNF- by UCB and adult T cells. Intracellular staining was performed in parallel and on the same MNC during the primary Con A stimulation experiments for NFAT1 analysis by Western blotting. MNC from UCB or adult peripheral blood were left unstimulated or stimulated using Con A for 40 hours in identical conditions and analyzed for intracellular expression of cytokines by flow cytometry. At 0 hour (unstim), 18 hours, and 40 hours of stimulation, CD3⁺ cells were gated and intracellular cytokine expression in gated cells was plotted versus CD69 expression. Numbers in histograms indicate the percentage of CD3⁺/CD69⁺ cells expressing the cytokine. (A) Intracellular staining for IFN-γ. (B) Intracellular staining for TNF-.