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.. The statistical significance of the magnitude of responses between time points for each peptide was determined by comparing the normalized triplicate values for each peptide. The normalization at each time was done by subtracting the average of the six background replicates of the background controls to each of the triplicates of the peptide responses. p-values for comparisons between time points (Figure 2B and Figure 3B) were then generated simultaneously using Westfall�CYoung max-T correction as in DFR, www.selleckchem.com/products/Temsirolimus.html but again with a modified test statistic: T=(XT1??XC1?)?(XT2??XC2?)��pooled, (6) written here to include the aforementioned normalization. Here, ��pooled=(NT1?1)��T12+(NT2?1)��T22NT1+NT2?2, (7) with: NT1=NT3=3 (8) Strongly significant refers to a result with a p-value less than 0.05 and moderately significant to a p-value less than 0.10. 3. Results and Discussion 3.1. Overnight Resting of PBMC Samples Increases the Magnitude of the Response to the A2-CEF Pool To evaluate the impact of ON resting of PBMC samples on the responses measured by IFN�� ELISPOT testing, PBMCs from four different HLA-A*0201 individuals were tested for their reactivity to a pool of five HLA-A2.1-restricted viral peptides from influenza PD173074 clinical trial (Inf) virus, EBV and HCMV (A2-CEF; an A2-condensed version of the CEF pool, known as a reliable control for detecting CD8 memory T-cell responses in PBMCs) [21]). Different vials of PBMC samples were non-rested (0 h. resting) or rested for 18 h or 22 h before testing. Responses from all donor PBMC samples to the A2-CEF pool at all three time points (0, 18 and 22 h) were positive and strongly statistically significant when compared with background control wells (PBMC alone) (see Figure 2A, Figure 2B, left, and the representative images of the ELISPOT results in Figure 4A). Although qualitative detection, as a matter of statistical significance, would not change based on whether the cells MRIP were rested (the p-values for all of the subjects at the three time points are