Rat cd11b m1 70

MacBlue and MacGreen mice were transcardially perfused with 0.9% saline and brain were removed and digested at 37°C for 1 h in an enzymatic cocktail consisting of 1x HBSS, 50 U/ml collagenase D, 100 µg/ml Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride, 5 U/ml DNase I and 8.5 U/ml dispase. The tissue was then manually homogenised using a Dounce homogeniser. Myelin was removed after centrifugation of the homogenate on a 35% percoll (GE Healthcare) gradient. The resulting single cell suspension was incubated with 1 µg/ml anti-CD16/CD32 (Biolegend) before staining with a cocktail containing rat CD11b (M1/70 1∶500, Biolegend), rat CD45 (30-F11 1∶200, Biolegend) and rat F4/80 (BM8 1∶200, eBioscience). Flow cytometry was performed using a BD Fortessa and data was analysed using FlowJo V10 software.

Flow cytometry was performed to quantify inflammation in the mouse tails after tail surgery.³⁷ Briefly, single‐cell suspensions were obtained from a 1‐cm portion of the tail distal to the surgical site using a combination of mechanical dissociation and enzymatic digestion. Cells were stained with anti‐mouse monoclonal antibodies: rat CD45 (30‐F11; #103139), rat CD3 (12A2; #100203), rat CD4 (RM4‐5; #100536), Armenian hamster CXCR3 (CXCR3‐173; #126511), rat CD11b (M1/70; #101207), Armenian hamster CCR4 (2G12; #131211), mouse Ly‐6G (1A8, #127607),), rat CD86 (GL‐1; #105011) and rat CCR8 (SA214G2; #150309), rat F4/80 (BM8; #MF48004), Armenian hamster CD11c (N418; #45‐0114‐82) from BioLegend (San Diego, CA, USA); Armenian hamster CCR5 (7A4; #12‐1951‐82) from eBioscience (San Diego, CA, USA).
For cell sorting, mouse tail skin was harvested 2 weeks after tail surgery and digested into single‐cell suspensions. Three different population (CD45+CD11b+, CD45+CD4+, CD45–) were obtained with BD FACS Aria™III (BD Bioscience).