Isolation and Characterization of Immune Cells from Rat Brain Post-Stroke

Twenty-four hours after stroke, the rat brain was isolated and subjected to mechanical and enzymatic dissociation using a tissue dissociation kit (Miltenyi Biotec), as previously described [33 (link), 34 (link)]. Suspensions of single cells were mixed with a Percoll suspension and centrifuged at 950 g for 30 min at room temperature. Cells were resuspended in PBS containing 2% BSA and were incubated with the respective antibodies at 4 °C:anti-CD45 (#202207; Biolegend, San Diego, CA,USA), anti-CD11b (#201805; Biolegend, San Diego, CA,USA), and anti-TGR5 (ab72608; Abcam Cambridge, MA,USA). BD CellQuest Pro software (5.1) (CA, USA) was used to determine immune subpopulations. Cells were then gated for CD45-high and CD45-intermediate populations. An Alexa Fluor®488-labeled donkey anti-rabbit IgG secondary antibody was used for the final detection. Data were analyzed using Flow Jo 7.6.1 software (Tree Star, US).