TRPM2-deficiency affects DC maturation. A) BMDCs were generated in vitro from WT and TRPM2−/− mice. Representative dot blot graphs indicating the percentage of DCs at d 6 of culture are shown. Samples were stained with APC-CD11c and eFluor 605-CD11b antibodies. B) Time course of DC proliferation of WT and TRPM2−/− BM cultures. C) BMDCs were matured by addition of TNF-α or CpG DNA, and the expression of maturation molecules class-II, CD86, CD80, and CD83 was analyzed by FACS. Changes in the maturation markers were assessed by comparison of TRPM2+/+ BMDC histograms (black line) and TRPM2−/− BMDC (red line) and histogram differences (green line). Statistical significance of the differences was calculated by applying the χ(T) or PB test, wherein a value T(X) > 4 implies that the two distributions are different, with P < 0.01 (99% confidence). T(X) values for TNF-α-treated DCs: class-II, 565; CD80, 990; CD86, 412; and CD83, 379. T(X) values for CPG-treated DCs: class-II, 68; CD80, 1072; CD86, 499; and CD83, 495. Data are from 3 independent experiments (n=3). D) Splenic cell suspensions were prepared from WT and TRPM2−/− mice, and samples were stained as in A. Representative dot blot graphs indicating the percentage of CD11b+CD11chi and CD11b+CD11cint DC populations and their total number. E) Splenic samples were stained as in C. Histograms represent mean fluorescence intensity (MFI) of MHC II, CD80, CD86, and CD83 in CD11b+CD11chi (solid bars) and CD11b+CD11cint (open bars) DCs; n = 10. Paired Student's t test was applied for significance analysis; P < 0.05 was considered statistically significant.