I have been culturing MEF cells and tried to use different transfection methods to transfect them and they show very low efficiency and we would like to make sure that they are indeed MEFs so that I can rule out this doubt and focus only on the transfection issue.....Thank you!!!
This thread looks interesting for you: https://www.researchgate.net/post/IHC-fibroblast_staining-what_marker_to_use
If you're not sure of the identity of the cell line, it is rather straightforward to generate new MEF cell lines. You also might want to think about how interesting it is to do experiments in a single MEF cell line of questionable identity. Better practices might involve performing multiple biological replicates with 3 or 4 MEF cell lines all from the same genetic background and immortilized in the same manner.
Primary cells including MEFs are difficult to transfect, I would recommend you try nucleofection (Lonza) or neon transfection system (Life technologies). To ensure your cultured cells are indeed MEFs you can stain with surface markers such as CD44 or Thy1.
Every MEF line differ in the transfectability (even from the same mouse line). Our mefs are very hard to transfect and now we have shifted to retroviral vectors. So I dont think based on this problem, u can doubt ur cell line. If they have fibroblast morphology, with high F-actin and vimentin, they should be fibroblasts.
In my opinion, if you prepare primary mefs (or even immortalize them), its very rare that anythiing else will grow better and overpopulate your culture.
Good Luck with transfection standardization as Aalam suggested above
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