Can you let me know about any resources for gating? I am new to gating and I'm not sure how to gate on lymphocytes population in one plot and get to different sub types of t cells in other plots. Please let me know how to gate in any dot plot figure. Thank you.

I can understand why u used FMO for CD25 (external marker) but I don't understand why you did not use Isotype for the intracellular staining because for intracellular staining some FoxP3 antibodies might bind unspecifically to internal proteins. Usually intracellular staining I use an ISO flourochrome and the surface staining I use FMO. If you use isotype for your intracellular staining and FMO for your surface staining your gating will look a lot better. You can try it. That is why your FoxP3 FMO plot is so low... You need an isotype so that your cells move more positive a bit.

Thank you! Now I start work with human FoxP3 and i have a big priblem with losing cells after permibialization. Can you help me with protocol washing cells?