Adult brain workflow - 
Isolation and analysis of neural cells

The cell suspension from adult brain tissue contains a significant amount of cell debris (A) and erythrocytes (B), which hampers subsequent cell isolation, cultivation, and analysis. The Adult Brain Dissociation Kit optimizes the tissue dissociation process, and after sample clearing yields a high amount of pure and living cells with much less cell debris (C) and erythrocytes (D).

A single-cell suspension from a 3 month 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 on the MACSQuant Analyzer.

A single-cell suspension from a 3 month old mouse brain was prepared using the Adult Brain Dissociation Kit, mouse and rat. Subsequently, adult neurons were isolated from the single-cell suspension with the Neuron Isolation Kit, mouse. Cells were fluorescently stained with antibodies specific for non-neuronal cell marker and the ACSA-2 antigen and analyzed by flow cytometry using a MACSQuant Analyzer. 

Isolation of mouse microglia using the CD11b MicroBeads, human and mouse from 2 month old mouse brain tissue. The cell separation using the MicroBeads resulted in a high purity of 98.8% and a viability of 95.2 %. Cell were stained with CD11b-FITC and CD45-APC for further analysis.

Single-cell suspension of adult brain tissue was used for cell separation with Anti-O4 MicroBeads, human, mouse, rat. Cells were stained with Anti-O4-PE to verify a pure cell population of pre-oligodendroytes. After separation, 92% of cells in the positive fraction were positive for O4.

Primary adult neurons were cultured in MACS Neuro Medium, MACS NeuroBrew-21, 1% P/S, 0.5 mM L-glutamine and BDNF for 7 days after isolation from adult brain tissue. Cells were fixed and stained with anti-MAP2 (green) and anti-βIII Tubulin (red) antibodies and DAPI (blue). 

Adult astrocytes were isolated from 8-week old mouse brains using the Adult Brain Dissociation Kit and the Anti-ACSA-2 MicroBeads. When using the AstroMACS Separation Buffer and the AstroMACS Medium, growth of astrocytes is strongly improved (A) in comparison to astrocyte culture without the AstroMACS Separation Buffer (B). Cells were fixed and stained with GLAST Antibody (green) and DAPI (blue).

Single-cell analysis of non-sorted adult neural cells led to identification of 24 different subclusters, representing neuronal and different glial cell types, including astrocytes, microglia, oligodendrocytes, vascular cells, and immune cells.

Isolation of neurons using the Neuron Isolation Kit resulted in an increased resolution of neuronal clusters, including several subpopulations of immature and mature neurons. Moreover, the amount of non-neuronal cell types was significantly decreased. 

Using the Adult Neural Stem Cell Sorting and Analysis Kit, mouse resulted in a pure population of neural stem cells from the subventricular zone. Cultivation of these neural stem cells led to the formation of neurospheres (A, B) and differentiation into neuronal and glial cells (C-F). Cells were fixed and stained with antibodies against GFAP (green), Nestin (red), GLAST (green), MAP2 (green) or O4 (green).