FACS Isolation of Astrocyte Subtypes

Mice were sacrificed at 2 weeks after surgery with a lethal dose of tribromoethanol (i.p., 500 mg/kg, Sigma-Aldrich, Darmstadt, Germany). The blood was washed out through intracardiac perfusion with cold 0.1 M PBS. Spinal cords were dissected, washed in 0.1 M PBS and transferred in 750 uL of PBS. Mechanical trituration was followed by enzymatic tissue digestion through 30 min incubation at 37 °C in the following solution: hyaluronidase 7 mg/mL, trypsin 13 mg/L, kynurenic acid 4 mg/mL (all from Sigma Aldrich, Saint Louis, MS, USA), DNAse I 10 mg/mL (Roche, Rotkreuz, Switzerland), and Mg2⁺ 25 mM (Sigma Aldrich, Saint Louis, MO, USA). Single-cell suspension was filtered on a 40 µm sieve (BD Biosciences, Franklin Lakes, NJ, USA), the sieve was washed with 0.1 M PBS and the solution was centrifuged at 700× g for 5 min. Supernatant was resuspended using 0.9 M sucrose in PBS and centrifuged at 700× g for 20 min to discard myelin fragments. Cells were incubated in mouse anti βIII-tubulin in PBS (1:100; R&D Systems, Minneapolis, MS, USA) for 20 min on ice. Cells were centrifuged for 5 min at 400× g, washed with cold 0.1 M PBS and incubated for 15 min on ice in Allophycocyanin (APC)-conjugated donkey anti mouse in PBS (1:100; Jackson Immunoresearch, Carlsbad, CA, USA). As controls, we used wild type mice (to show cell distribution without any staining), uninjured mice (to show eGFP expression alone), wild-type mice stained with βIII-tubulin (to show APC staining alone), and without primary antibody (Figure S2). Cells were centrifuged (5 min at 400× g), washed with cold 0.1 M PBS and re-suspended in 7-AAD before sorting. We used BD FACSAria™III. (Becton Dickinson cell sorter BD Biosciences, Franklin Lakes, NJ, USA) equipped with 638 nm laser (670-14) and 561 nm laser (610-20) and controlled using FACSDiva software (Becton Dickinson cell sorter BD Biosciences, Franklin Lakes, NJ, USA). Size threshold was used to eliminate debris. Two cell populations were sorted simultaneously: non-converting (Aldh1l1eGFP⁺/βIII-tubulin⁻) and converting astrocytes (Aldh1l1eGFP⁺/βIII-tubulin⁺). Number of mice included in the FACS experiment: 30 Aldh1l1-EGFP hemisected male mice.