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Flow cytofluorometry analysis - (Aug/27/2005 )

I am looking for some advice about how to analyse data from flow cytometry.
I am trying to measure changes in regulation of a protein using cytofluorocytometry. Have have successfuly fluoro-tagged my protein and I am able to get a reading above background from the cytometer. I am not sure however the best way of subtracting the background fluorescence from the fluorescence of my protein so that I have meaningful measurement to compare between conditions. Most papers seem to use a qualitative statement to describe the level of fluorescence above background, whereas I really need quantitative data.

Any help would be much appreciated. smile.gif


There are a few ways you could probably do this (I assume you're using the BD Cellquest software and flow cytometers, although I guess the generally gist should be the same). Are you expressing your protein by stable or transient transfection ?

If the protein is stably transfected you could use histogram statistics to give you mean fluorescent intensities (mfi) for your non-expressing and expressing cells and subtract the values to get a value for the expression level.

Overlaying the expressing histogram onto the non-expressing histogram and gating on expressing cells will give you a percentage of cells expressing detectable levels above background.

If only a low percentage of cells are likely to be transfected you could specifically gate out these cells, using an M1 gate and your non-expressing cells and look at changes in mfi or the percentage of cells expressing in this way.

Try a few ways and see what looks best. For absolute expression mfi is a good bet. For looking at populations of cells or an all or nothing situation, percentage of positive cells is useful.

Good luck,


Dear Rob,

FACS analysis is quite complicated and getting good meaningful data from it depends to a large degree on how you setup the machine, for example forward scatter, side scatter, gain, compensation...

Gates and quadrants would probably also help you. However I doubt that any explanation I write here will make sense to you. I strongly suggest that either you attend a FACS training course, get a colleague to train you properly (not just show you how to switch the machine on) or at the very least read some good training literature.

This might help if you are using CellQuest (program comes with the Becton Dickinson FACS machines):

If you are using a different make/program, is a god place to start. Maybe check out the website of the supplier company?