I have isolated granulosa cells from mice ovary using puncture method. Cells are healthy and growing in DMEM. Their morphology is same as granulosa cells. However I would like to use a molecular markers or Immunohistochemistry method to authenticate them further, before downstream use. Can anyone suggest the best methods and markers for the same ?
Dear Dr. Vani Khare
You may use Immunocytochemistry (ICC) to further characterize the granulosa cells which you have isolated from mice ovary. In ICC, the staining technique is applied on cultured cells that have been isolated from tissues or blood samples. On the other hand, in immunohistochemistry (IHC), cells are analyzed within intact tissue sections.
You may use forkhead box L2 (FOXL2), which is a key ovarian transcription factor expressed mainly in pregranulosa/granulosa cells.
I have attached a few references which may be helpful.
https://academic.oup.com/biolreprod/article/86/2/37,%201-9/2530631
Article Functional Oocytes Derived from Granulosa Cells
https://www.cell.com/cell/fulltext/S0092-8674(09)01433-0?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867409014330%3Fshowall%3Dtrue
Article Etiology of Ovarian Failure in Blepharophimosis Ptosis Epica...
Article The murine winged-helix transcription factor FOXL2 is requir...
https://pubmed.ncbi.nlm.nih.gov/15944199/
https://www.nejm.org/doi/10.1056/NEJMoa0902542?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200www.ncbi.nlm.nih.gov
https://www.novusbio.com/products/foxl2-antibody_nbp2-22473
Regards,
Malcolm Nobre
Malcolm Nobre thank you for detailed answer with all the references. it's really helpful. I was reading about the FOXL2 too, and wondering if I should use this as marker instead of FSHR or Cyp19 as their expression can change based on the stage. Your answer is very convincing. I will give FOXL2 a try. Also what's your opinion on ICC vs flow cytometry - which one is more acceptable as the granulosa cell purity test from the publishing POV ?
Dear Dr. Khare,
I had flow in mind too. When it is between ICC vs flow cytometry, you should opt for flow cytometry as per your requirement.
Best.
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