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A workflow protocol to purify neural stem cells (NSCs) from wildtype mouse brain. This entire experimental setting resulted in highly pure (>95%) and viable NSCs (>90%) in less than 3 h.
First, an optimized automated dissociation protocol is applied, which ensures high viability and epitope integrity of the resulting single cell suspension. Then, NSCs are identified by detection of the exclusion markers CD24, Ter-119, and CD45 and the NSC specific markers GLAST and PlexinB2. Subsequently, purification of NSCs is carried out with the MACSQuant® Tyto® Cell Sorter. A neurosphere assay can be performed to verify the viability and functionality of the sorted NSCs.
The MACSQuant Tyto Cell Sorter is a multi-parameter cell sorting device that uses a micro-chip based sorting technology for sterile and gentle cell isolation. Unlike conventional droplet sorters, cells do not experience high pressures and no charge is applied, ensuring high viability and functionality.
Coat a 24-well culture dish with 0.01% Poly-L-lysine overnight at 37 °C. Wash three times with ddH₂O the following day.
Prepare the following cell culture medium: MACS Neuro Medium containing 2% MACS NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine.
Use the Neural Tissue Dissociation Kit (T) to dissociate brain tissue and prepare a single-cell suspension. Follow the protocol of the kit data sheet.
Download data sheet
Isolate the GLAST-positive astrocytes from the single-cell suspension using the Anti-GLAST (ACSA-1) MicroBead Kit. Follow the protocol of the kit data sheet.
Download data sheet
Mouse forebrain cells before separation
GLAST (ACSA-1)-negative cells
GLAST (ACSA-1)-positive cells
Effective isolation of GLAST+ cells from mouse brain tissue. Mouse brain tissue (P7) was dissociated using the Neural Tissue Dissociation Kit (T), the gentleMACS™ Dissociator, and FcR Blocking Reagent, mouse. Astrocytes were isolated from single-cell suspension using the Anti-GLAST (ACSA-1) MicroBead Kit, a MiniMACS™ Separator and an MS Column. Cells were fluorescently stained with Anti-GLAST (ACSA-1)-APC and analyzed by flow cytometry on the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
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Copyright © 2019 Miltenyi Biotec and/or its affiliates. All rights reserved.