Isolation, characterization, and cultivation of astrocytes

Cell suspensions from adult brain contain a significant amount of cell debris and erythrocytes (A), which hampers subsequent cell isolation, cultivation, and analysis. The Adult Brain Dissociation Kit, mouse and rat optimizes the tissue dissociation process, and after sample clearing yields living cells with much less cell debris and erythrocytes (B).

Mouse whole brain tissue (P4) was dissociated using the Neural Tissue Dissociation Kit (P) and the gentleMACS™ Octo Dissociator with Heaters. The cells were stained with Propidium iodide and analyzed with the MACSQuant® Analyzer. Flow cytometry analysis showed that the dissociated cell suspension contained minimal debris (A) and a very high percentage of viable cells (B).

Isolation of neonatal astrocytes. Single-cell suspensions from P3 mouse brain tissues were prepared using the Neural Tissue Dissociation Kit (T) (A) or Neural Tissue Dissociation Kit (P) (B). Neonatal astrocytes were isolated from the single-cell suspensions using the Anti-GLAST (ACSA-1) MicroBead Kit, human, mouse, rat (A), or the Anti-ACSA-2 MicroBead Kit, mouse (B). Cells were fluorescently stained with Anti-GLAST antibodies (A), or Anti-ACSA-2 antibodies (B) and analyzed with the MACSQuant Analyzer.

Isolation of adult astrocytes. A single-cell suspension from 3 months old mouse brain was prepared using the Adult Brain Dissociation Kit, mouse and rat. Adult astrocytes were isolated from the single-cell suspension with the Anti-ACSA-2 MicroBead Kit, mouse. Cells were fluorescently stained with the Anti-ACSA-2 antibody and analyzed by flow cytometry using the MACSQuant Analyzer.

Neonatal astrocytes were isolated from P4 mice using the Neural Tissue Dissociation Kit (P) and the Anti-ACSA-2 MicroBead Kit. Cells were cultured in MACS® Neuro Medium and MACS NeuroBrew®-21 (top) or in AstroMACS Medium (bottom) at different densities in a 24 well imaging plate for 7 days. Cells were fixed and stained with Anti-GLAST antibody
(yellow) and DAPI (blue) and dead and viable astrocytes were analyzed with the Cytation™ Cell Imaging Multi-Mode Reader. (A) Microscopy images of one representative well. (B) Quantitative data of 4 replicates.

Neonatal astrocytes were isolated from P4 mice using the Neural Tissue Dissociation Kit (P) and the Anti-ACSA-2 MicroBead Kit, and cultured in AstroMACS Medium at a density of 10,000 cells / well in a 24 well imaging plate for 7 days. Cells were then fixed and stained with Anti-GLAST antibody (green) and DAPI (blue).

Adult astrocytes were isolated from the brain of 8-week-old mice using the Adult Brain Dissociation Kit and the Anti-ACSA-2 MicroBead Kit in combination with (A) or without (B) the AstroMACS Separation Buffer. Cells were then cultivated in AstroMACS Medium at a density of 100,000 cells/well in a 24-well Imaging Plate for 7 days. Cells were fixed and stained with Anti-GLAST antibody (green) and DAPI (blue).

PFA-fixed cultured astrocytes from neonatal mouse brain (A) and cryosections of PFA-fixed adult mouse cerebellum were stained with Anti-GLAST pure (red), Anti-ACSA-2 pure (green), and DAPI (blue); and analyzed by confocal microscopy. ACSA-2 is co-expressed with GLAST in the cerebellum, but showed a brighter expression in the granule cell layer. A detailed staining protocol is available for download on the right.