To me, the smaller population looks like apoptatic cells. I assume your live/dead cells marker is for necrotic cells which have permeable membrane for this dye, but the membrance of the early apoptatic cells is intact, they are shrinking, and show more particles inside, all this match your description of that population.  Meanwhile, there always are some activated T cells will die after strong stimuli.

 To validate this guess, you can include anther dye, say annexin V for early apoptosis.

 If i am right,  i don't think you should include these cells for your function work if you are not working on apoptosis.

Dear Pedro, I agree with Dr Shiqiu Xiong. When I analized ex vivo isolated splenocytes from mice immunized with MHC class I disparate tumor, cells in this area showed enhanced staining with Annexin V.

I aggree with my fellow researchers, they look like death cells. Which is not stange after an aggressive activation like we usually use in vitro with anti cd3/anti cd28.

I would suggest that these are cell which are ready to go. and it looks  that anti Cd3 and Cd28 stimuli is not giving the synchronized cell activation and division. If it is the case then if you should centrifuge the plate after adding the Cd8 cell, these cell will be deviding  along with other cell and you will not see this population. Another case if you stimulate for longer time then this small population will disappear but in this case you will lose the activated population by AICD.

So you should synchronize the culture after stimulation

Thank you guys! Shiqiu Xiong's explanation makes sense. I won't include them in my analysis.

Cheers

 I am a student and usually read most of these responses. They are very helpful, thanks a lot.

I am currently doing my project, and I would be grateful if anyone could help me understand the concept of "pulsing DCs with a peptide antigen"

Particularly, how does T cells proliferate when introduced to the pulsed DCs when there is no antigen processing and hence (I suppose) no expression of co-stimulatory molecules.

Thank you.

Hi Nana,

My understanding of pulsing DCs with peptide is that the DCs, being phagocytic cells. will "swallow-up" the peptide, process it and present it on their own MHC molecules. Alternatively, its possible that simply by adding an overwhelming amount of peptide to the culture and these peptides have high-affinity for a specific MHC that they will replace the peptide that is currently being presented.

Whatever the case, the DC will now present your peptide and offer a signal I to the T cell, together with signal 2 (co-stimulation, particularly if you activated the DCs previously with LPS) and possibly signal 3 (IL-2, IL-12, etc).

Perhaps someone else has a more accurate idea of what is going on.

Cheers

P