After removing germ cell contamination by hypotonic treatment with tris, Myoid cell contamination is still present in the cell culture.So is there any other treatment by which myoid cells contamination can be minimized or removed??
I would suggest the use of FACS. Sertoli cells express SOX9 uniquely. Exploit this surface marker. You can check out this publication:
Cell Transplant. 2002;11(6):499-505.
Cells isolated using FACS may not be appropriate for functional studies, as common fluorochromes (e.g., propidium iodide) used for cell sorting are toxic.So we need a technique which can circumvent this problem.
The person who has downvoted my answer I request you to please give me an appropriate answer....:P
How long and at which temperature do you incubate the cells with Tris? What is your Tris anyways?
I seed the Sertoli cells onto Datura stramonium-coated cover slips. After incubating the cells for 1 h at 35°C and 5% CO2, I have been using 0.3x HBSS for 3-5 min. It works quite well.
We work with Sertoli cells primary cultures since many years. To remove peritubular myoid cells we performed a 1M glycine-2mM EDTA (pH7.4) treatement during the isolation procedure. At day 5 of culture, no myoid cell contamination was detected when an immunoperoxidase technique using a specific antiserum to alpha smooth muscle actin was applied. For details of Sertoli cell isolation procedure you can see Meroni et al. J. Endocrinol. (2002) 174:195-204.
Eliana Thanks a lot for your help,but can you tell me the exact time and temperature for which 1M glycine-2mM EDTA (pH7.4) treatement should be given? Previously we have given the treatment for 10 mins and incubated at room temperature with normal agitation.
The treatment should be carried out for 10 minutes at room temperature, during this period we performed only gently sporadic pipeting.
- Badges
- Science topic