Albumin ovomucoid inhibitor solution

Four male P5 nonIR and IR cerebella were dissected into ice-cold 1× HBSS (Gibco) and were pooled for downstream analysis. Two replicate experiments were performed. All the steps were performed on ice when possible. Cerebella were minced with a clean blade and then dissociated in Accutase (Innovative Cell Technologies) at 37°C for 10 to 15 min. Following dissociation, Accutase was washed out using neural stem cell media (Neurobasal, supplemented with N2, B27, and nonessential amino acids, Gibco). Following filtering through a cell strainer and trituration in media to single cells, cells were layered over a 5-ml density gradient (albumin-ovomucoid inhibitor solution, Worthington) and centrifuged at 70g for 6 min to remove debris. The cells were briefly treated with 1:2 1× red blood cell lysis buffer (Sigma-Aldrich) for 2 min at RT. Cells were washed twice (500g, 5 min at 4°C) and resuspended in 1× tris-buffered saline. Cells were stained with calcein acetoxymethyl live stain dye (1:500; Thermo Fisher Scientific) on ice for 30 min and were passed through a 40-μm cell strainer to remove any cell clumps before loading onto a microwell device.