Approximately 3 million cells per brain were prepared for flow cytometric analysis. Viable cells were labeled with LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen). Samples were washed with PBS and treated with Fc Block to inhibit nonspecific binding (BD Biosciences, Sparks, MD). Cells were then labeled with FITC Ly6c (AL21), PE Cy7 CD11c (HL3), Alexa Fluor 700 Ly6G (1A8), and APC Cy7 CD45 (30-F11). A common channel for PE-CF594 CD3 (145-2C11), CD19 (1D3), Siglec F (E50-2440), and NK1.1 (PK136) was used (BD Biosciences). Additionally cells were labeled with PerCP Cy5.5 MHCII (M5/114.15.2), PE CD64 (x54-5/7.1) (Biolegend, San Diego, CA), Alexa Fluor 647 F4/80 (BMA) (Abdserotec, Raleigh, NC), and efluor450 CD11b (M1-70) (eBioscience, San Diego, CA). Following incubation, all cells were washed with PBS and fixed with 1.6% paraformaldehyde.
Flow cytometry data were acquired on a BD LSR II cytometer (BD Immunocytometry Systems, San Jose, CA) having 405-nm, 488-nm, 561-nm, and 640-nm excitation lasers as previously described by our laboratory (20 (link)). Data collection was performed using BD FACS Diva Software (BD Biosciences), and data analyses were performed using FlowJo Software (Treestar, Ashland, OR). Bioexponential transformation was adjusted when necessary.
Flow cytometry data were acquired on a BD LSR II cytometer (BD Immunocytometry Systems, San Jose, CA) having 405-nm, 488-nm, 561-nm, and 640-nm excitation lasers as previously described by our laboratory (20 (link)). Data collection was performed using BD FACS Diva Software (BD Biosciences), and data analyses were performed using FlowJo Software (Treestar, Ashland, OR). Bioexponential transformation was adjusted when necessary.