Characterizing Adult Subependymal Zone Cells

Characterization of NSC and NPC populations in the adult SEZ was performed as previously described [38 (link), 39 (link)]. Roughly, SEZ tissue was dissected, minced and enzymatically digested using the neural tissue dissociation kit (T) (Miltenyi, 130–093-231) in a gentleMACS Octo Dissociator with heaters (Miltenyi). After trypsin inhibition, digested pieces were mechanically dissociated, the cell suspension was filtered through a 40 μm and treated with the Dead Cell Removal Kit (Miltenyi, cat no. 130-090-101) following the instructions of the manufacturer. Finally, the eluted living fraction was pelleted (300×g, 10 min) and incubated with the specific cocktail of primary antibodies [38 (link), 39 (link)] (1:300 CD24-PerCP-Cy5.5, BD 562360; 1:100 CD31-BUV395, BD 740239; 1:200 CD45-BUV395, BD 565967; 1:20 CD9-Vio770, Miltenyi 130-102-384; 1:20 GLAST-PE, Miltenyi 130-095-821; 1:30 O4-Biotin, Miltenyi 130-095-895; 1:200 Ter119-BUV395, BD 563827; 1:300 AF488 EGF complex, Molecular Probes E13345) and reagents (DAPI, 50 µg/ml) at 4 °C for 30 min. Labeled samples were analyzed using a LSR-Fortessa cytometer (Becton Dickinson) with 350, 488, 561 and 640 nm lasers.