Fig. 4
Flow cytometric phenotyping of microglia using pro- and anti-inflammatory DAM markers. a Selection of potential flow cytometric markers for Magenta (pro-inflammatory) and Yellow (anti-inflammatory) DAM modules in acutely isolated WT and 5xFAD microglia. Criteria included kME ≥ 0.75 for one module, ≤0.5 for the other module and predominant cell membrane localization. Yellow module: Cxcr4 (CD184); DAM-phenotype: CD11c and Pdcd11 (PD1); Magenta module: CD44, CD274 (PDL1), CD45 and Kv1.3. Cell surface functional Kv1.3 channel expression was detected by ShK-F6CA, a fluorescein-conjugated Kv1.3 channel blocker that selectively binds to Kv1.3 channels. b Acutely isolated CNS immune cells were gated for live cells (FSC/SSC) and then for single cells (using FSC-A/FSC-H) followed by CD11b positivity. CD11c was used as a marker of DAM based on prior RNAseq studies. CD11c+ DAM were gated as shown and proportions of CD11c+ DAM in CD11b+ CNS immune cells were compared between WT and 5xFAD mice (n = 6 mice/group, age 6–8 mo). c Comparison of pro-inflammatory DAM (CD44, CD45 and CD274) and anti-inflammatory DAM (CXCR4) specific markers within CD11c+ DAM in 5xFAD and WT mice (n = 6/group, age 6–8 mo). Grey histogram represents isotype control. d Flow cytometric phenotyping of CD11b+ cells in mouse brain based on CD44 and CXCR4 expression. e Changes in proportions of DAM subsets in aging WT and in aging 5xFAD mouse brains (n = 4 mice/group/time point). Pairwise and group-wise comparisons and significant differences are highlighted. Levels of significance: *p < 0.05, **p < 0.01, ***p < 0.005. Colored markers indicate within group comparisons (Red: 5xFAD, Blue: WT) while Black markers indicate inter-group differences (WT vs. 5xFAD)