Cell population analysis of the adult murine subependymal neurogenic lineage by flow cytometry

Abstract

Summary This protocol provides a flow-cytometry-based procedure to classify and isolate all cells of the adult rodent subependymal zone (SEZ) neurogenic lineage, without the need for reporter mice, into different cell populations, including three neural stem cell (NSC) fractions with molecular signatures that are coherent with single-cell transcriptomics. Additionally, their cycling behavior can be assessed by means of 5-ethynyl-2′-deoxyuridine (EdU) incorporation. Our method allows the isolation of different NSC fractions and the functional assay of their cycling heterogeneity and quiescence-activation transitions. For complete details on the use, execution, and outcomes of this protocol, please refer to Belenguer et al. (2021) .

The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2

“…Consistent with this, SEZ homogenates showed a 40% increase in cell yield (Fig. 5c); however, when we scored specific cell populations by flow cytometry [38,39], we found that the increase in NSCs (CD24 −/low GLAST + CD9 high cells) was within the range of the generalized hyperplasia of the tissue, whereas NPCs (GLAST − CD24 −/low EGFR + cells) were significantly overrepresented with an increase of 70% in Cdkn1b mutant mice (Fig. 5c).…”

“…6 b–d). As with regard to the neuronal lineage, doublecortin (DCX) + early neuroblasts generated from NPCs can proliferate once or twice as they retain EGFR and are sensitive to mitogens before they exit cell cycle as late nondividing EGFR − neuroblasts that constitute, by far, the largest population in the SEZ neurogenic niche [ 2 , 38 ]. We found that DCX + neuroblasts in wild-type mice had very low or undetectable levels of SOX2 and ASCL1 [ 40 , 41 ] together with high levels of p27 (Fig.…”

“…Characterization of NSC and NPC populations in the adult SEZ was performed as previously described [ 38 , 39 ]. 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).…”

“…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 , 39 ] (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.…”

“…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 [38,39]…”