Isolation of Brain and Spleen Immune Cells

Isolation of immune cells from brain was performed as previously described [52 (link), 54 (link)]. Briefly, brain hemispheres were mechanically homogenized in dissection buffer (HBSS, Gibco™, Germany), supplemented with 50 mM glucose (Roth, Germany) and 13 mM HEPES (pH 7.3, Sigma), using a glass potter, and then filtered through a 70-μm cell strainer (Falcon®, Corning, Germany). The cell suspension was washed (400 g, 10 min, 4 °C) with PBS and fractionated on a discontinuous 30%–70% isotonic Percoll® gradient (GE Healthcare, Germany) (800 g, 30 min without brake, 4 °C). After the removal of myelin debris, cells in the interphase comprising mononuclear cells were isolated and washed with FACS buffer (PBS w/o Ca²⁺/Mg²⁺, 2% v/v fetal bovine serum (FBS), 10 mM HEPES, 0.1% sodium azide), centrifuged (400 g, 10 min, 4 °C) and immediately used for flow cytometric analysis. To isolate immune cells from spleens, organs were homogenized and sieved through a 40-μm cell strainer (Falcon®, Corning). Isolated cells from spleen and blood samples were treated with erythrocyte lysis buffer (eBioscience, Germany), and washed twice with FACS buffer (400 g, 10 min, 4 °C) before staining and flow cytometric analysis.