Hello, I am doing flow cytometry to evaluate the pluripotency of mouse embryonic stem cells (mESCs) which have been derived and cultured on mouse embryonic fibroblasts (MEFs)
Unfortunately, I am struggling with distinguishing between my mESC and MEF population only on the basis of size (FSC) and granularity (SSC). As I am using different pluripotency markers- Nanog (APC), Oct4 (APC) and Sox2 (PE), I was hoping to separate the mESC populations, given their pluripotent characteristics. As controls, I also stained MEFs with Oct4 and Sox2, and although they didn't show any fluorescence for SOX2, they stained positively for Oct4. I have also observed MEFs to be a little autofluorescent, so I am not sure if they are showing positivity for this marker, or it is a false positive.
The following paper shows that monolayer MEFs do express pluripotent markers such as Nanog, Oct4, Sox2 and SSEA-1, but I am a little confused if the mitotically inactive (irradiated and/ or mitomycin treated) MEFs would also express these markers?
(http://onlinelibrary.wiley.com/doi/10.1111/dgd.12043/pdf)
And if they do, could you please suggest ways to separate MEFs from mESCs for flow cytometry? I know these are much bigger cells, but my cells show a very mixed sized profile, which is making it quite hard to distinguish. I am relatively new to flow, and if you have any tips acquiring mESCs on flow, please do share.
I would very much appreciate any suggestions or inputs from your side.
Many thanks.
Pooja.
If you cannot use size, a quick and easy way to sort mESC from MEF by flow cytometry is SSEA1 which is a surface marker expressed only on ESC and not on MEF.
Dear Marie,
Thank you for your reply. I tried the cell surface marker SSEA-1, but my staining didn't work as much. Could you please share the surface marker staining protocol for flow cytometry, and also if you have combined it with intranuclear staining as well.
Thank you.
Hello
Yes i agree with Marie , The MEF cell no showing any expression with the embryonic stem cell markers SSEA-1 and SSEA-3
See links here hope help you
Good Luck
http://www.pnas.org/content/113/1/122.full
Article Mouse Embryonic Fibroblasts (MEF) Exhibit a Similar but not ...
Hi Pooja, I am currently having the same issue. I culture mESCs with feeder mEFs and then stain for Sox2 (APC), OCT4 (FITC), and Nanog (PE). However, when I'm analyzing the data I obtain a dual population (singlets vs doublets). I was analyzing the singlets as mESCs and found them to be negative for Sox2 but positive for Oct and Nanog. It then occurred to me that maybe the "singlets" are the MEFs and the "doublets" are the actual stem cells. I have looked up some references stating that MEFs express pluripotency markers, but I am not sure as to why they would be Sox2 negative but Oct4 and Nanog positive in this case. I was thinking that the next step would be to stain for a fibroblast marker to be able to distinguish both cell types and do the correct analysis. Did you ever figure it out? Did you try staining for SSEA? Thanks! Pooja Khurana
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