Recent advances in genome editing and the application of fluorescent proteins have accelerated Interest in isolating specific populations of brain cells from mixed populations. Researchers are also using cell sorting to isolate single cells for expansion and analysis.
Follow these 5 guidelines to address the challenge of obtaining single cell suspensions from delicate brain tissues.
- The methods chosen to dissociate brain tissue for obtaining single cells should be gentle and impart low shear stress in order to preserve cell surface antigens and cell viability.
- The desired region of the brain is dissected and the relevant tissue is minced.
- Cells are further isolated through enzymatic digestion using papain or Accutase with EDTA.
- Large debris and cell clusters should be removed by filtering through mesh prior to staining and sorting.
- Viability dyes such as DAPI should be included to monitor cell death. The number of dead cells can be used to determine consistency and quality of cell preparation.
Several studies have been published in which scientists have successfully isolated stem cells from whole brain homogenate using cell sorting. The references below provide more detailed information about preparing and sorting brain tissue.
- Daynac, Mathieu, Lise Morizur, Thierry Kortulewski, Laurent R. Gauthier, Martial Ruat, Marc-André Mouthon, and François D. Boussin. "Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics." Journal of Visualized Experiments: JoVE (103 (2015). https://www.ncbi.nlm.nih.gov
- Wylot, Bartosz, et al. "Isolation of vascular endothelial cells from intact and injured murine brain cortex—technical issues and pitfalls in FACS analysis of the nervous tissue."Cytometry Part A 10 (2015): 908-920. http://onlinelibrary.wiley.com
- Guez-Barber, Danielle, et al. "FACS purification of immunolabeled cell types from adult rat brain."Journal of neuroscience methods 1 (2012): 10-18. https://www.ncbi.nlm.nih.gov