V500 anti mouse cd45

Mice were euthanized through inhaled anesthetic and perfused with phosphate-buffered saline (PBS). Brain and spinal cord tissues were harvested and enzymatically dissociated with collagenase (MilliporeSigma, St. Louis, MO, USA) and DNase I (Roche, Basel, Switzerland) and subsequently mechanically dissociated by vigorous pipetting. Leukocytes were enriched by Percoll (MilliporeSigma) density gradient centrifugation. Cells were treated with Fc blocker (BioLegend, San Diego, CA, USA), stained with the indicated anti-mouse antibodies for 30 min at 4 °C [V500 anti-mouse CD45 (BD Bioscience, San Jose, CA, USA), APC anti-mouse CD11b (eBioscience, San Diego, CA, USA)], and analyzed by flow cytometry. CNS-derived cells were stained and analyzed individually for each mouse. Live cells were determined by forward and side scatter fluorescence on a BD LSRFortessa X-20 Cell Analyzer (BD Bioscience). Flow cytometry data analysis was performed using the FlowJo software (FlowJo, Ashland, OR, USA).