Hi all, I've just found something interesting about autofluorescence in flow cytometry.
I've found, that autofluorescence, especially on FL1 [ex 488 / em 533] and FL2 [ex 488 / em 585] channels is increased in unhappy (stressed, or damaged by centrifugation, or hypoxic, or dying -?), but still whole cells. There are 3 clear peaks on FL1 and FL2 hystograms:
- the peak with the lowest fluorescence are cell debris with very low FSC and SSC.
- the middle peak are apparently healthy cells
- the far-right peak are apparently damaged cells, with somewhat lower average FSC and higher average SSC than healthy cells.
I've checked in the internet, and have read, that reduced NADH contributes to autofl. on FL1, and oxidized FAD - to FL2, and that increase of autofl. at these two channels may indicate that the cells experience hypoxia.
Would be nice to read comments of experienced people on this issue.
Autofluorescence depends on a lot of other, pretty much unknown and hard-to-control-for parameters. It is thought that chemicals with aromatic rings and altering single and double covalent C-C bonds are the main contributors to this phenomenon.
It might be true that these compounds you mention contribute to AF, but AF is definitely not good for the EXACT SCIENTIFIC MEASUREMENT of hypoxia - there are a lot more other, widely published and accepted methods for that. Moreover, the autofluorescence rates of different cells and tissues vary a lot.
Regards, Domokos
Autofluorescence depends on a lot of other, pretty much unknown and hard-to-control-for parameters. It is thought that chemicals with aromatic rings and altering single and double covalent C-C bonds are the main contributors to this phenomenon.
It might be true that these compounds you mention contribute to AF, but AF is definitely not good for the EXACT SCIENTIFIC MEASUREMENT of hypoxia - there are a lot more other, widely published and accepted methods for that. Moreover, the autofluorescence rates of different cells and tissues vary a lot.
Regards, Domokos
Lipofuscin is also a big source of autofluorescence. If you are doing flow cytometry with mouse cells I suggest you use young animals for your experiments.
Good luck
Jaime Llodra
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