Fig. 4

ABT-263 treatment decreases proportion of microglia in spinal cord. A) Experimental design demonstrating induction of EAE followed by ABT-263 treatment at symptom onset and spinal cord flow cytometry at 18 d.p.i., and at least 4 days post-treatment start. B-D) Cell gating parameters for evaluation of viable single cell population. E) Gating strategy for isolating CD45-Cd11b- cells (R1) from CD45 + cells (R2). F) Division of R2 into three cell subsets, consisting of CD45 high Cd11b positive innate immune cells (R3), CD45 mid Cd11b positive microglia (R4) and CD45 + Cd11b- lymphocytes (R5). G) Division of R3 by Ly6C and Ly6G fluorescence to differentiate monocytes/macrophages (Ly6C-Ly6G-) from other innate immune cells (Ly6C + proinflammatory monocytes, and Ly6G + neutrophils). H) Division of microglial population based on Ly6C and Ly6G positivity demonstrating most cells (> 85%) in the R4 microglial population are negative for both. I) Division of the R5 lymphocyte population by CD4 and CD8 positivity. J) Quantification of the percent of viable cells within the innate immune cell population (R3 + R4) population demonstrating a significant decrease in population in ABT-263 treated animals. K-M) Division of the R3 population (CD45 high Cd11b+) based on Ly6G (K) and Ly6C (L) positivity, and the R3 population negative for both (M), demonstrating significant decrease only in the Ly6G and Ly6C negative population. N) Evaluation of the microglial subset in vehicle treated and ABT-263 treated mice as a percent of total viable cells, demonstrating a significant decrease in treated mice in both the whole population. O-Q) Analysis of the R5 lymphocyte population (O) based on subdivision of CD4 (P) and CD8 (Q) positivity showing no change in the number of CD4 + or CD8 + lymphocytes present in the spinal cords. * < 0.05, all comparison’s conducted with one-tailed unpaired t-test, vehicle n = 3, ABT-263 n = 4