We digested the brain tissue with Trypsin ( 1x) for 15 min (all steps were performed in cold conditions) and suspend the cells by pipetting up and down (3-4 times) to the single-cell suspension. We evaluated the percent of apoptosis, necrosis and live cells using flow cytometry. The result of flow cytometry was found that the percentage of cells in the necrotic phase was enhanced. How can I decrease the necrotic cells?
Treatment with trypsin may cause this kind of necrosis. Try to use neutral solutions for digestion. Anionic or Cationic solutions are reported to cause cell damage. Go for Accutase instead of trypsin.
Attaching file for different homogenization methods.
Treatment with trypsin may cause this kind of necrosis. Try to use neutral solutions for digestion. Anionic or Cationic solutions are reported to cause cell damage. Go for Accutase instead of trypsin.
Attaching file for different homogenization methods.
May I ask in which cell type are you interested in?
In our lab for example we isolate microglial cells from adult brains using the Gentle MACS from Miltenyi Biotec and then use the cells for FACS analysis. This protocol works quite good. You can isolate other cell types like astrocytes, olygodendrocytes or neurons.
https://www.miltenyibiotec.com/DE-en/applications/neuroscience.html
You don't necessarily need to isolate a particular cell type, but instead you use the first part of the protocol to just dissociate the tissue and remove the debris and blood cells.
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