This application protocol uses the Anti-GLAST antibody to generate highly purified and viable astrocytes from neonatal mouse brain tissue. A single cell-suspension is generated from brain tissue of mice younger than P8 through combined enzymatic and mechanical dissociation. Astrocytes are isolated from the single-cell suspension using the Anti-GLAST (ACSA-1) MicroBead Kit. This procedure was tested particularly on P5–7 dissociated mouse brain tissue, derived from CD-1® mice and containing approximately 12–18% GLAST+ cells.
PB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA) by diluting MACS® BSA Stock Solution 1:20 with PBS. Keep buffer cold (2−8 °C). Prepare fresh on the day of use and degas the buffer, as air bubbles could block the column.
▲ Note: BSA can be replaced by other proteins such as mouse serum albumin, mouse serum, or fetal bovine serum (FBS).
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.
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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.
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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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|Column||Max. number of labeled cells||Max. number of total cells||Separator|