Help with my FACS histograms, please - (Aug/06/2007 )
As the pictures, left one is the ctrl, right one is the EGFP test.
Can the results define a EGFP positive population?
Why the mean fluorescence intensity of the positive population is so low?
Or a false positive result? 
Thanks!!
hi,
from this figure, it appears that 2 populations of cells exist in the EGFP test tube
for the control, peak is at 2e2 (100% of the cells)
for the EGFP, a second peak is at 8e2 (for approx. 60% of the cells)
it is true that the shift in fluorescence is not drastic, but it is significant (4 fold the intensity of the ctrl!)
it depends on the level of expression of the GFP protein...
what about the apparent mortality of the cells in this test?
Sebastien
from this figure, it appears that 2 populations of cells exist in the EGFP test tube
for the control, peak is at 2e2 (100% of the cells)
for the EGFP, a second peak is at 8e2 (for approx. 60% of the cells)
it is true that the shift in fluorescence is not drastic, but it is significant (4 fold the intensity of the ctrl!)
it depends on the level of expression of the GFP protein...
what about the apparent mortality of the cells in this test?
Sebastien
Hello all-
Sebastein,
Would you at all suspect aggregate protein for a lower shift in fluorescense?
from this figure, it appears that 2 populations of cells exist in the EGFP test tube
for the control, peak is at 2e2 (100% of the cells)
for the EGFP, a second peak is at 8e2 (for approx. 60% of the cells)
it is true that the shift in fluorescence is not drastic, but it is significant (4 fold the intensity of the ctrl!)
it depends on the level of expression of the GFP protein...
what about the apparent mortality of the cells in this test?
Sebastien
Thank you sebastien
What is the apparent mortality? I've never examined this parameter before.
Could you please tell me from where(i.e. books, references, sites, and etc) I can learn to analyse FACS results?
Dear Bradley,
Your control cells should really be in the first decile (0-10) and there is no need to have 10to6 displayed in your case, as your cells aren't that bright. Adjusting gains and voltages could potentially lead to a better discrimination between the populations, but you definitly have a shift.
I really would recommend a training course for using the FACS machine, software and analysis package. There's simply no way you'll understand it from reading books or papers - I've tried that myself. Only thing to do is to go to a practical course. Not sure where you are in the World, so I can't possibly recommend anything. But the Purdue University Cytometry website should give you a good idea.
Best wishes
LeserattePD
from this figure, it appears that 2 populations of cells exist in the EGFP test tube
for the control, peak is at 2e2 (100% of the cells)
for the EGFP, a second peak is at 8e2 (for approx. 60% of the cells)
it is true that the shift in fluorescence is not drastic, but it is significant (4 fold the intensity of the ctrl!)
it depends on the level of expression of the GFP protein...
what about the apparent mortality of the cells in this test?
Sebastien
Thank you sebastien
What is the apparent mortality? I've never examined this parameter before.
Could you please tell me from where(i.e. books, references, sites, and etc) I can learn to analyse FACS results?
Thank you so much, LeserattePD