I am currently trying to dissociate normal human lung tissue that has been frozen @ -80C for about three weeks to set up for MACS sorting and upon my first round I obtained an extremely low number of cells (~250k) in single cell suspension. This is my first time dissociating tissue that has been frozen but I am running a second round today with more tissue and slightly longer incubation periods for dissociations (I perform them serially with human tissue, e.g. incubate for 25 mins in dissociation solution, pull off remaining tissue into a new conical and incubate for another 25 mins and combine the two single cell suspensions at the end). I have attached my rough protocol that I put together for this. I thaw the tissue by swirling in our 37C bead bath quickly and transferring the tissue to a petri dish and washing with ~5mL DMEM/F12 + 10%FBS. I then centrifuge and aspirate and repeat the wash. If anyone has successfully done this process and can point out where the discrepancy may be, that would be greatly appreciated.
Update: Got 4.5 million on the second round. Still would like to see a better yield
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