Hello, I have isolated cancer stem cells from MCF-7 cells. I want to characterize these cells. Is there any option available other than FACS? Can we do it with PCR or western blot for markers of CSCs?
There's no reason you COULDN'T use PCR or Western Blot, other than not having the right reagents just now. Western Blot is very similar to FACS analysis - both use antibodies to specifically label the proteins, and if an antibody exists for FACS, chances are somebody sells one for the same protein that works well in Western blotting applications. FACS will simply tell you what's happening with each individual cell, while Western Blot gives only the average of your total population (unless you use some of the fancier single-cell devices that have come out recently). PCR is a different measurement entirely - it looks at genetic material rather than protein - but if you design the right primers, a qPCR could give you the relative amounts of substantially more characteristic markers than either blot or FACS would in a single run.
You can go for immunofluorescence and check for the presence of pluripotency markers like oct4, sox2 stc. or cancer stem cell markers like CD44, CD133.
Dear Heena,
once you have isolated a putative cancer stem cell population you should immediately perform your analysis for additional markers either indicating a stem-like state or a more differentiated state. This analysis could be done either via additional flow cytometry which is a senisitive method which also allows you to combine different cell surface markers e.g. expression of CD133 and CD44. With qPCR the analysis could be much broader and even more sensitive, however this would be RNA level. I would start with qPCR and than validate some interesting/important markers via immunofluorescence or FACS or western blot. But keep in mind that cancer stem cells may show a very high cellular plasticity, meaning that the cell population you have isolated via FACS may undergo a phenotype switch within some days of culturing. Hence the best way would be to directly use sorted cells for addtional analysis or RNA extraction. sorted cells can be stored frozen as pellets at -80 °C.
Hey Heena, maybe this summary can give you some insights..
https://www.promocell.com/blog/characterization-mesenchymal-stem-cells-poster/
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