I am working on male mice. I have the difficulty to culture sertoli cells from adult mice. As sertoli cells no. become fixed in adult. I used percoll gradient method and use lectin but was not able to find sertoli cells, would some one grace me by solving my problem?
Please be specific your problem your not getting cells after isolation or else with protocol...let me knw the protocol first. I will help you
after chopping the testis I digest it in 10ml of DMEM F-12 enriched collagense 1A (0.5µg/mL)250µl and DNase I (200µg/mL) 400µl along with shaking and at 36C for 20 mints and then pour on 40ml of 5% percoll in HBSS in 50 ml tube. keep it for 1 hour at 37C in incubator. select bottom 5ml portion in 10ml of DMEM, DNAse and 4ml of trypsin for 20 mints digestion same as above. halt the reaction by adding 4ml of FBS enriched media, mix and pass through 200 no (70µm) sieve then wash in DMEM/HBSS twice. dissolve the pallet in media, place in lectin DSA added petri dish(@ 5µg/mL) which is already prepared 1 hour before and stored at 37C. before use I wash the lectin added dish with HBSS twice. store it for 1 hour or more sometime at 37C. then replace with new FBS enriched media at 37C in 5%CO2 incubator. next day i wash the dish and change media. sometime I use 0.3X HBSS to remove the germ cells. but still i get no good result.
HBSS1x no cal, no meg. (gibco)
0.25% Typsin EDTA 1x (gibco)
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