Question - FACS analysis (Feb/25/2011 )

Hello All,

I have a question about FACS samples. If the samples have about 10% and 90% separated that is 90% of population away from vertical 'y' axis and the small 10% close to 'y' axis, (both higher up from horizontal 'x' plane), then do we assume the sample has 2 populations? Or just ignore the smaller group and gate the 90% population for our analytical understanding purpose?
My understanding is that the smaller population might be dead cells or debris or non specifically bound AB during staining. I am sorry I cannot post the picture because my computer does not let me to. But I hope I explained my doubt clearly. And yes, the FL2 peak was quite narrow and good.
However, I gated the 10% population too and found that it showed good expression of FL2. As I told I assumed that it might be cell debris bound to AB during staining or dead cells bound during AB staining.
Others argue, even if it did not show in the peak as 2 separate peaks, it is still 2 populations. What is the explanation to this?

Thank you and regards,
Scicel

10% 90% separation is on FSC vs SCC? or FL1 vs FL2?

I think if you look at FSC vs SCC, you should be able to tell whether it is cell debris or not
(I mean if the comp etc is fine)

if the separations is on FL1 vs FL2, then gate each population and look at thier distribution on FSC vs SCC

Hi Thanks for your reply.

10% 90% separation is on FSC vs SCC. The FL1 peak was uniform and shows a single narrow peak which makes it look like a single clone. But the question was raised by one of our scientist who says since the FSC SCC shows slight seperation, we cannot consider it as a single clone and the argument was that maybe a small percentage of second population is mixed.
Cell debris is at the bottom right? This small population was slightly above but seperated from the 90% population. For the FL1 I usually gate the bottom most (almost the all the dead cells and Include everything except that small gated (dead) population. (I suppose you understand that this kind of gating includes everything in our analysis except the dead cells!!) Again this kind of gating analysis came from the same scientist (which I am ok) but how to explain the small population of 10%?

Thank you,
Scicel

Rnotk on Mon Feb 28 07:09:27 2011 said: