CFSE based FACS analysis

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biplabbose
biplabbose's picture
CFSE based FACS analysis

We are doing cell proliferation assay using CFSE staining, followed by FACS analysis as per the protocol published in Nature Protocols.
We are measuring the fluorescence intensity of CFSE stained cells in FL1 channel of the FACS. But am bit confused whether to collect FL1-A data or FL1-H data . As I understand one should collect FL1-A data to avoid the effect of change in cell size during cell cycle.
However, I do see many publications where they have collected FL1-H data and have shown that as a histogram. Most of the publications simply put it as “CFSE intensity”, with out saying FL1-A or FL1-H.
It will be nice if you can give some advice on this.

samm
samm's picture
FL1-A data is more accurate,

FL1-A data is more accurate, but many older, non-digital machines (BD FACStar or FACScan with Cell Quest acquisition) acquired data in 'H' parameters by default - so much of what you see in older papers might be inertia. New digital acquisition systems can store the full set of data, so you can compare A&H parameters. The only place I utilize H in CFSE staining now is as the initial doublet discriminator - after that its the fl-A parameters across 5 channels to actually analyze populations.
Also, the area parameter helps normalize noise and gives better stats, whereas height does not, but both parameters do measure CFSE intensity.
If you think your separations aren't wide enough (i.e. doubling factor is not 0.5), try reducing the amount of CFSE.

biplabbose
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Thanks Samm!!!

Thanks Samm!!!