Hello,
I am isolating primary murine glial cells from different neonatal knockout strains (p0-p2). After several rounds (up to 4 times) of harvesting the microglia, I am dissociating the astrocyte monolayer from PDL coated 75 cm² flasks (containing 2 brains). At this step, I am having issues getting single cells, the astrocytes are sticking together and forming big chunks of cells.
Although I have aleady tried different methods of dissociation such as using different kinds of narrow pipettes, trypsine, DNAse, papain, cell strainers and so on, I am still getting big chunks of cells. The cells seem to be fine since live/dead staining with trypan blue indicates that the cells are pretty viable (less than 5% dead cells).
Does anyone have a clue what makes the astrocytes stick together after their dissocation from the cell flasks?
Thanks in advance!
Kind regards,
Laura
Hi Laura, I found an interesting paper tht compares different approaches for isolating cells from brain tissue, maybe it can help you. You might want to try change your reagents.
Article A non-aggressive, highly efficient, enzymatic method for dis...
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