Isolation and cultivation of mouse neurons and neuronal precursor cells

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 neurons from neonatal and adult mouse brain. (A) P1 mouse brain was prepared using the Neural Tissue Dissociation Kit - Postnatal Neurons and neonatal neurons were isolated from the single-cell suspension using the Neuron Isolation Kit, mouse. Cells were fluorescently stained with antibodies specific for non-neuronal cell antigens and analyzed by flow cytometry using the MACSQuant Analyzer. (B) Brain tissue from 3-months-oldmice  was prepared using the Adult Brain Dissociation Kit, mouse and rat and 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 ASCA-2 antigen and analyzed by flow cytometry using the MACSQuant Analyzer.

Primary adult mouse neurons were cultured in MACS Neuro Medium, MACS NeuroBrew-21, 1% P/S, 0.5 mM L-glutamine and BDNF (incubation for 3–6 hours with 50 μg/ml BDNF at day 3) on PLL-coated glass coverslips. After 7 days cells were fixed and stained with the neuron-specific antibodies Anti-MAP2 (green) and ßIII Tubulin (red).

PFA-fixed cultured neuronal cells and cryosections of PFA-fixed cerebellum and olfactory bulb were stained with CD171, pure, mouse antibody and analyzed by fluorescence microscopy. A detailed staining protocol is available for download on the right.

PFA-fixed cultured neuronal precursor cells and cryosections of the PFA-fixed hippocampus were stained with Anti-PSA-NCAM, pure, human, mouse, rat antibody and analyzed by fluorescence microscopy. A detailed staining protocol is available for download on the right.