CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The <t>PBMC</t> and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, <t>non-cryopreserved</t> PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.