For both treatment groups, cortices from individual animals were collected separately from four LPS-injected or saline-injected control animals. The hippocampus, cerebellum, and olfactory bulbs were removed, as were the meninges. For the MCAO model, the ipsilateral cortex was collected, while the contralateral cortex, and other brain regions were discarded. Four mice that had undergone MCAO and four sham-operated control surgery were used. Dissected tissue was treated as described previously12 – briefly, dissected tissue was first diced to 1–3 mm and then digested with 200 U of papain enzyme for 90 min at 34 °C in bicarbonate-buffered Earle’s balanced salt solution with 0.46% glucose, 26 mM sodium bicarbonate, 0.5 mM EDTA, and 125 U/ml DNase I (Worthington Biochemicals). Digested tissues were dissociated into single-cell suspensions by gentle trituration, and myelin removed using O4, Mog, and GalC supernatants (1:30 at room temperature for 30 minutes). Myelin and larger tissue clumps were removed by filtering through 3 layers of Nitex mesh, and cells collected by centrifugation, before resuspension in Dulbecco’s PBS (DPBS) containing 0.02% BSA and 125 U/ml DNase I and with LIVE/DEAD® Fixable Far Red Dead Cell Stain Kit (ThermoFisher, L34973) for fluorescence-activated cell sorting (FACS).
