I am trying to isolate neural stem cells from the spinal cord of 6 month old mice. I'm washing the sample 4 times with 1X PBS + 2% P/S and 1% Amphotericin. Dissociating into small pieces and then for 25 minutes in Tryple Express. Inhibiting enzyme action with DMEM High Glucose + 10% SFB, filtering on 40um cell strainer and culturing in DMEM/F12 1:1, 2% B-27, 1% L-Glutamine 200mM, 1% MEM Non-essential aminoacids, 20ng/ml EGF, 20 ng/ml FGF-2 into a 25cm² bottle. After 4-7 days of culture, these are the structures I observe and they do not look like neurospheres, but like cell clumping.
you may filter cell suspension thru 70um filters, twice to get clean preparation of single cells,
Second culture in low binding flasks or also called low adhesion flasks,
You should get spheres in a week
good luck
Subhash C. Juneja thanks for replying! I was filtering only once in 40 um cellstrainer. This was being a bit difficult because of the amount of tissue blocking the filter pores. I will test your recommendation and filter in 70 um instead. Thanks for the tip!
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