Fluorescence and Fixing

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Fluorescence and Fixing

Inga Koneczny

I am trying to establish a flow cytometry experiment and found something weird that I can not explain.

I transfect (HEK293) cells with a construct expressing dsred in an IRES, but when I fix the cells in 1%PFA, the fluorescence is strongly reduced, almost disappears, compared to unfixed cells.

With the same construct - the only difference is, instead of Dsred there is GFP in the IRES- I repeat this experiment, and upon fixing the fluorescence of my cells is enhanced. (The median fluorescence is a lot higher in fixed vs unfixed cells.) But I do not see much of an increase in autofluorescence in untransfected cells.It also does not look like stronger auto-fluorescence when I look at my histograms.

Does anybody have an idea how 1% PFA (made from 36% stock, diluted with medium, made fresh at least 1x per month) could have such a weird impact on fluorescence?

Best regards,
Inga (1st year DPhil student)

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