This application protocol describes a step-by-step protocol to isolate pure neural stem cells (NSCs) from the subventricular zone (SVZ) of adult mouse brain. Brain tissue from adult mice was dissociated into single-cell suspensions using the Neural Tissue Dissociation Kit (T) in combination with the gentleMACS™ Dissociator with Heaters. After dissociation, cell debris was removed using the Debris Removal Solution. NSCs were stained using the Adult Neural Stem Cell Analysis Cocktail Kit, anti-mouse and sorted using the MACSQuant® Tyto® Cell Sorter. The MACSQuant Analyzer 10 was used for flow cytometric acquisition and data analysis. Neurosphere cultivation and differentiation assays were performed afterwards using the MACS® Neuro Medium and MACS NeuroBrew®-21 w/o Vitamin A.
|Enzyme mix 1||Enzyme mix 2|
|Enzyme T||Buffer X||Buffer Y||Enzyme A|
|200 µL||1750 µL||20 µL||10 µL|
|Tissue||Debris Removal Solution||D-PBS||Overlay D-PBS||Reagent tube|
|20–100 mg||450 µL||1550 µL||2 mL||5 mL|
Positive collection chamber:
Negative collection chamber:
Dissociation of subventricular zone tissue yielded 3.69×105± 9.3×104 cells per mouse (7–9 weeks old) with a viability rate of >97% (n = 12), using the Neural Tissue Dissociation Kit (T) and gentleMACS™ Octo with Heaters. This figure shows a representative analysis of the different fractions after exclusion of debris, doublets, and dead cells. Sorting of 3.69×105± 9.3×104 total cells resulted in 36,000 ± 8,000 GLAST+ Plexin-B2+neural stem cells with a purity of >95% and a viability rate of >93% as analyzed by propidium iodide staining (n = 6).
Figure 5: Analysis of CD24, CD45, and Ter119 expression.
Figure 6: Analysis of Plexin-B2 expression.
Figure 7: Analysis of GLAST (ACSA-1) expression.
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