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?