Dissecting and Analyzing Mouse Brain Cells

Normal brains of adult Ang-2 GOF mice and wild-type littermates were dissected and immediately transferred in ice-cold HBSS. Briefly, following gentle mincing, the tissue was incubated in an HBSS solution containing Collagenase P (0.2 mg/ml), Dispase II (0.8 mg/ml), DNase I (0.01 mg/ml), Collagenase A (0.3 mg/ml) for 60 min at 37 °C under gentle rocking as previously described [52 (link)]. Following myelin removal, the cells were preincubated with rat anti-mouse FcγIII/II receptor (CD16/CD32) blocking antibodies (≤1 μg/million cells/100 μl; BD) for 5 min at 4 °C, and stained with the fluorochrome-conjugated antibodies (0.25–1 µg): CD45 PercP Cy5.5 (clone 30-F11, BD Biosciences), Gr-1 APC-Cy7 (clone RB6-8C5, BD Biosciences), CD11b APC (clone M1/70, BD), F4/80 FITC (clone BM8, eBioscence). Following two washing steps, DAPI (4′,6-diamidino-2-phenylindole; Invitrogen) was added for live gating and cells were acquired on a FACSCanto™ II flow cytometer (BD) using Diva Software (BD) and further analyzed using FlowJo analytical software (FlowJo Version 10.0.8, LLC). Background fluorescence levels were determined by Fluorescence Minus One (FMO).