Which population to gate? - FACS gating question (Jan/18/2008 )

Hi, everyone, I isolated splenocytes from 6 week old naive B6 mice (no peptide immunization) and cultured cells in 24-well plate 15*10^6 cells/ml medium/well in the presence of a mixed peptide (Copaxone, a mixed peptide designed for treatment of Multiple Scleorosis) for 4 days without addition of IL-2.

Here is my medium: 1640+10% heat-inactivated FBS+50uM beta-ME+10mM Herps.

4 days later I analyzed cells by FACS. Unfortunately, i got 5 populations instead of 1 (I usually got 1 in the case of fresh
splenocytes) in the SSC-FSC window. Then, i gated every region and saw the expression of CD4. Here is the result.

My question is:

Which population should I gate?

Why?

Can anyone tell me exactly what kind of cells each region means?

Is IL-2 necessary for naive T cell/peptide culture?

Just to clarify a bit...I wrote that last post in a rush as I was sooooooooo excited that my husband got a new great job!.....

I am just assuming that you have a mixture of myeloid and lymphoid cells as you have only cultured the cells for 4 days ex vivo. But I don't know what effect your culture system has on the cells. Have you also FACS splenocytes that were cultured without the peptide? When you look at splenocytes FSC v SSC from normal mice I always see that G3/4/5 population. The bigger cells (ie: higher FSC and SSC would be the myeloid cells). And the smaller cells are the lymphocytes. Did you stain the cells with PI prior to FACS to exclude dead cells? That would give you a cleaner FACS and perhaps explain why all your cells are CD4+ (it should only be the T lymphocytes!). I hope this helps