I am doing multicolor flow cytometry analysis on stromal cells from the human intestine, and a disproportioned amount (70-80%) of my “Stromal” cell population (live, CD45-, EpCAM-, CD31-) cells are not staining for any of seven different markers (such as CD146, CD34, PDGFRa), so what could they be?
The cells are obtained from biopsies or small amounts intestine, and basically prepared by first removing epithelial cells using media containing EDTA, and then enzymatic digestion of the tissue. Even though these “naked cells” are seen mostly towards the lower end of the FSC values, they overlap the FSC and SSC of the Live, CD45+ population (as well as other populations), suggesting that at least not all of the naked cells are debris. Moreover, the markers do work and stain varying amounts of the remaining cells. So what are these naked cells? And can I get rid of them?
It's difficult to say what you are seeing. One issue is that the cells were liberated by enzymatic digestion - what enzyme(s) and for how long? - which may have cleaved antigens off the surface. Without knowing all the antigens you tested, and not knowing their susceptibilty to proteolytic cleavage, it is impossible to speculate on what type of cells you are seeing in your FACS analysis.
Hello,
You can make single cell suspension by mechanical disruption of cells by either wire-mesh or cells strainer or frosted glass slide (without enzyme). Try this method, if some problem in enzymatic disruption than it can be handled.
Thanks for the advise Gary, they have been noted. And to you to Mohammad, I might use your suggestions some other time, but changing the enzyme buffer (as well as small tweeks to the protocol) did the trick.
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