Hello Maria,
PMA and ionomycin stimulation results in non-specific activation of all your CD4+ T cells (they act directly on the protein kinase C pathway). As a result of this strong stimulation, CD4 expression (as well as CD3) gets downregulated by the cell - so you won't get the same plot of CD4 expression when compared to unstimulated.
One way of gating on these cells is to take the whole CD4 population (if plotted against CD8s) but you will of course include some cells that will not be true CD4s. Alternatively, you can do intracellular staining of CD4 but if you do surface and intracellular stains it will result in using 2x more antibody. Although I never tried myself, I know some people surface stain for CD3 and CD4 prior to stimulation with non tandem antibodies (PE, APC etc).
I only use this condition as a positive control for IFNy and IL2 production so surface staining followed by gating on CD4Lo cells does the job.
I hope this helps.
Aleks
Hello Maria,
PMA and ionomycin stimulation results in non-specific activation of all your CD4+ T cells (they act directly on the protein kinase C pathway). As a result of this strong stimulation, CD4 expression (as well as CD3) gets downregulated by the cell - so you won't get the same plot of CD4 expression when compared to unstimulated.
One way of gating on these cells is to take the whole CD4 population (if plotted against CD8s) but you will of course include some cells that will not be true CD4s. Alternatively, you can do intracellular staining of CD4 but if you do surface and intracellular stains it will result in using 2x more antibody. Although I never tried myself, I know some people surface stain for CD3 and CD4 prior to stimulation with non tandem antibodies (PE, APC etc).
I only use this condition as a positive control for IFNy and IL2 production so surface staining followed by gating on CD4Lo cells does the job.
I hope this helps.
Aleks
Agree with above -following stimulation I've been able to easily gate on the CD4 population by staining for CD3, CD4 and CD8 - gating on CD3 positive and then CD4 and CD8 - the CD4 staining is lower than in unstimulated cells but easily seen and can be differentiated from CD3 DN cells. I stain after stimulation and do not routinely stain for intracellular CD4 although this works
best
carolina
Maria-
I routinely stimulate human PBMC with PMA/Iono as a positive control for cytokine production. I agree with the other comments in that both CD3 and CD4 are internalized after this stim. I've never stained surface before stimulation, but rather stain intracellular CD3 and CD4 along with the cytokines I'm interested in. I had to test numerous clones & fluor combinations to find those that worked after fixation and the washing with perm buffer that is required after intracellular staining. As a place to start, check Cytometry Part A for OMIP articles which are highly optimized multicolor flow panels, including intracellular staining protocols.
Good Luck!
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining