Protocol for isolation and culture establishment of adult murine neural stem cells using tissue grinder and dissociator-based mechanical dissociation.

Here, we present an enzyme-free workflow for isolating murine adult neural stem cells (aNSCs) using tissue grinder- and dissociator-based mechanical dissociation (TIGR-MD). We describe dissection of the dentate gyrus and subventricular zone, mechanical tissue dissociation, cell recovery, and establishment of primary neurosphere cultures. We further provide procedures for neurosphere maintenance, passaging, and validation. TIGR-MD avoids enzymatic exposure during initial tissue dissociation and supports standardized establishment of primary murine aNSC cultures.