Hello! I am trying to stain CD4+ T cells for IL-17A and RORyt with no luck. I've been using the Foxp3/Transcription factor staining set from eBiosci and while the IL-17 staining looks *strictly fine* , the RORyt is always negative.
my IL-17A is APC and RORyt is BV605 if that helps.
Has anyone had any success with this staining?
If you are having trouble finding a positive population you can first verify the antibody is working by staining CST beads.
You then would want to ask yourself if RORyt will be highly expressed in healthy cells? Some receptors/proteins require priming before you will see any noticeable separation from a negative population. Do you need to stimulate cells with IL-6 before you stain for flow?
Are you trying to quantify RORyt, or use it as a gating strategy?
Lewis Williams Thank you for the reply. I am using RORyt as a gating strategy to quantify Th17 cells in a population of lung and spleen cells. Do you have a recommendation for stimulation including types, amounts, and times you use?
One issue you may be encountering is insufficient permeabilization of the nucleus, giving you enough to detect cytosolic targets but not enough to stain nuclear targets. The eBio kit is one of the best for simultaneously detecting cytokines and transcription factors, so if you’re using the manufacturer’s protocol this probably isn’t your issue.
You might check your antibodies, try a different antibody lot or stain a comp bead and check that they’re still working. APC and BV605 are fairly stable and bright fluores so that shouldn’t be an issue. I’ve had difficulty in the past with our core using suboptimal filters for BV605 detection, so if you’ve had issues with this channel in the past you might look into that. Are you losing too much of your BV605 signal to spectral overlap limiting your resolution? Transcription factors can have poor separation compared to cytokines or surface proteins so FMO-defined gating can help you get the best resolution. Also, double check that your RORyT antibody is reactive to the appropriate species.
With CD4 T cells, you normally don’t need to stimulate cells to detect the lineage-specific transcription factors but you usually do need a stimulation step + golgi inhibitor to get cytokines to detectable levels. If I understand your prior comment, it sounds like you did not a stimulation step but still saw good separation of IL-17? Not impossible, but surprising.
For stimulation I typically use 1.6 ug of agonistic CD3 antibody and 2 ug of agonistic CD28 antibody bound to a flat-bottomed 96-well plate. PMA/Ionomycin stimulation is more commonly used in the literature. For either stimulation, I do it 5 hours at 37C in the presence of BFA + Monesin (golgistop and golgiplug from BD using their protocol) and stain for my targets. There are a lot of variations that people use for this protocol but I’ve had good luck staining T cells under these conditions.
Hope this helps and good luck
Brett P Gross Thank you for the advice! I will look into the channels and filters on the machine to see if that is the problem. I will also try using BFA prior to staining -- the IL-17A separation without stimulation may have been a isolated occurance because I haven't been able to reproduce it.
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