Hello, I am planning an experiment to detect human antigen-specific TH2 T cells and would like you to learn from your previous experience. By using flow and ICS, in a time frame of 6h or overnight Ag (fungal peptides in my case) stimulation, which cytokine is easier to detect : IL-4, IL-5 or IL-13? Any hints will be appreciated!
Thanks for your help.
Marcelo
I agree with Boris Tartakovsky
that you will need to test to find out. From a technical perspective, several of these are difficult (if even possible) to measure depending on the species you're working with. For example, measuring any of these from nonhuman primates will be a challenge.I would add that I have seen some researchers combine some of these in the same channel to maximize sensitivity. For example, if the presence of any of these three markers is enough to define "Th2", you could use antibodies for all three which are coupled to the same fluorophore. You won't be able to decipher which cytokine is being expressed, but you'll be able to increase your chances of seeing a response and increase the number of cells that express any of these. All three of these can be dim and tricky to detect, so I would suggest coupling any and all of them to your brightest fluors like PE, BV421, and APC. Also, I highly suggest titrating these, as too much antibody is going to diminish your ability to see these signals. Check out Activated VeriCells sold by BioLegend to help titrate/optimize your panel.
Dear Marissa, thank you for your answer and this very interesting idea of combining antibodies for all 3 cytokines on the same fluorescence channel!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology