Hi,
I am wondering why our positively-selected CD8 T cells became two population on FSC-SSC plot (as shown below) after activation by using Dynabeads?
click the square next to the color. so the yellow and red cells are the cells you want and green or blue cells are others.
While activating CD8 T cells with anti-CD3/CD28, they should enlarge/blast.
Having control without stimulation should help at figuring it out.
Another possibility that often happens with beads is that it could represent T cells still attached to beads. It could also represent doublets of cells if you did not select singlets.
Hi Kaifeng,
it looks like your question did not get fully answered yet. As a note, it would be helpful to know which timepoint post stimulation your plot represents (1 day? Multiple days?), but basically the answer to your question is two-fold:
First, unless you remove the beads from your culture, you will actually see the beads on your cytometer when running the sample, i.e. the lower population probably contains your stimulation beads.
Second, some of your cells will die during the stimulation and as a result change scatter, and typically these dying cells will shift to being FSC-low (not all though, which is why it is essential to have a live-dead marker).
So the population that is higher on FSC are your T cells. You would expect to have some blasts as well, so make sure that you include the FSC-SCC high cells as well.
Good luck with your experiments,
Florian
We also observed this phenomenon after stimulation of CD4 T-cells with immobilized anti-CD3 and anti-CD28 antibodies (no beads are attached!!!). We believe that enlargement is to be expected due to the activation of the T cells as cytosolic protein synthesis and gene transcription increase. By just gating on enlarged T cells you might discover that their activation profile is more pronounced (cytokine expression or expression of activation markers).