Good afternoon, lovely people of ResearchGate.
In my assay on ImageStream, my unstained samples (e.g. when creating compensation matrix) have quite strong positivity in several channels (the strongest in channel 7, 435-505nm). I tried with both lysed full blood and with isolated PBMCs, the cells were fixed with 4% PFA or 4% FA (tried both with same result), washed with PBS + BSA then measured. This doesn't happen to such an extent in unfixed cells.
Anyone have an idea what could be causing such high fluorescence in unstained cells and how to avoid it?
Thanks a lot
Adam
What is your cell of interest? Are you discriminating between lymphocytes, monocytes, and granulocytes-is the background fluorescence different between cell types? Is the PFA freshly prepared? Do you see this autofluorescence on a traditional flow cytometer? Is someone using DAPI on the machine right before you????
Dear Matthew,
I was looking at monocytes.
In the meantime, I learned that your last question was the most important one - indeed our machine didnt flush sufficiently in between samples. Once I started incubating + washing away the Hoechst, the fluorescence went away.
Three days lost, but lesson learned :)
Still, many thanks for answering!
Adam
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