hi everyone,
I've been trying culturing neural progenitor cells (NPC) from dentate gyrus of adult (6-8w) mice but always failed to get neurospheres in serum free conditions. I can't find where is the problem, so please help me:
here is my procedure:
1) dentate gyrus dissection (from 2 brain) and cut into pieces;
2) digest tissue blocks with trypsin for 20 min at 37deg;
3) stop digestion by adding serum and remove the supernatent after a short centrifugation;
4) add the culture medium (DMEM/F12 with L-Glu, 20ng/ml EGF, 20ng/ml bFGF, 1%N2 suplement) and pipette the digested tissues with 1ml tips about 15 times to obtain single-cell suspension;
5) filter the suspension through cell strainer (40um) and centrifugate at 2000 rpm for 5mins and remove the supernatent;
6) add culture medium and suspend the deposition and centrifugate again;
7) resuspend cells with medium and count;
8) seed cells in 6-well plate and change the medium every 2 days;
can anyone give me a mature protocol on the culture of neural progenitor cells from adult dentate gyrus? thank you very much!
I'm waiting for your reply!
best regards
Xiangchun
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