Cell isolation

Microglia for the ischemic versus control (data in Fig 1) were isolated from the brain of CX3CR1cre^ERT2‐ROSA26 tdTomato (tdT) mice via FACS after intracardiac perfusion with 2 ml of cold PBS. Brains were collected in cold HBSS buffer (w/o Ca²⁺ and Mg²⁺; #14175‐053, Thermo Fisher Scientific). The brain tissue was enzymatically dissociated using the Neural Tissue Dissociation Kit (P) (#130‐092‐628, Miltenyi Biotec). The gentleMACS™ Dissociator (#130‐096‐427, Miltenyi Biotec) was used for mechanical dissociation steps following the Neural Tissue Dissociation Kit (P) manufacturer protocol for dissociation without heaters. The digested tissue was filtered twice with 70 μm and 40 μm filters, washing with cold Hanks' balanced salt solution (HBSS; with Ca²⁺ and Mg²⁺; #14025‐092, Thermo Fisher Scientific). Cells were separated from myelin and debris by 30% isotonic percoll gradient (#17‐0891‐01, GE Healthcare) in Myelin Gradient Buffer (MGB). Samples were centrifuged at 950 × g without acceleration or brake, for 30 min at 4°C. Cells were collected from the bottom of the tube, washed once with cold FACS Stain Buffer (#554656, BD Biosciences), and processed for FACS in a FACSAriaII sorter (BD Biosciences). No staining was required since the expression of tdT was used to separate microglial cells.

Microglia for the old versus young comparisons were isolated from the brain of WT mice after the assigned depletion/renewal protocol and ischemia. Mice were euthanized under deep anesthesia and the brain was processed in the same way as for the flow cytometry experiments to obtain a single‐cell suspension (see above). Unspecific binding was blocked by incubation for 10 min with anti CD16/CD32 (in FACS buffer at 4°C). Live/dead Aqua cell stain was used to determine the viability of cells. Cells were incubated with the following primary antibodies during 30 min at 4°C: CD11b (clone M1/70, APC‐Cy7, #557657, BD Pharmingen), CD45 (clone 30‐F11, FITC, #553080, BD Pharmingen). When sorting included Bodipy staining, as in flow cytometry, we used Viakrome 405 Fixable Viability Dye (#{"type":"entrez-nucleotide","attrs":{"text":"C36614","term_id":"2372755","term_text":"C36614"}}C36614, Beckman Coulter) instead of Aqua. After washing with FACS Stain Buffer (#554656, BD Biosciences), the cells were sorted in a FACSAriaII SORP sorter (BD Biosciences). Microglial cells were collected in sterile DPBS (#14190‐094, Thermo Fisher Scientific), centrifuged, and resuspended in lysis buffer (from PureLink™ RNA Micro Kit #12183016, Invitrogen) supplemented with 10% β‐mercaptoethanol and finally snap‐frozen in dry ice.