Flow cytometry

The method for preparation of BSL for flow cytometry has been reported. 17 Mice were sacrificed and perfused via the heart with 20 mL of perfusion fluid to remove blood contaminants from the cerebral vasculature. Brains were mashed between frosted glass slides in PBS and digested with collagenase type IV (Sigma, St. Louis, Missouri, USA) and DNase I (Sigma). Low‐density dead cells/debris/myelin were removed by resuspension in 30% Percoll followed by centrifugation. Cells obtained from the brain were suspended in 2% (v/v) FCS in PBS containing anti‐CD16/32 (2.4G2) (BD, New Jersey, USA) to block Fc receptors. Cells were labelled by incubating with fluorochrome‐labelled antibodies (see Supplementary table 1) diluted in FACS buffer for 30 min on ice after which cells were washed and stained with 0.5 μg mL⁻¹ DAPI (Invitrogen, Carlsbad, California, USA) for dead cell exclusion. Data were acquired on a FACSCanto II (BD), LSRFortessa (BD) or LSR‐II flow cytometer (BD) and analysed using FlowJo software (Treestar, Ashland, Oregon, USA).