Hello,
Does anybody here have experience with staining IL-10 receptor on T cells? Preferably for flow cytometry analysis.
I tried on splenocytes from a steady state mouse for 40min at 37°C or 4°C, but it doesn't show me any proper staining.
Any recommendations would be highly appreciated!
Best,
Valerie
You need to first set experiments to optimize time for IL10 and IL10-R. After specific stimulation, take few cells at different time points (depending upon your cell type it may vary from 5-6 hrs to 24 hrs) to stain with IL10-R expression. In such case published protocol (time and stimulation dose may vary due to different SOPs of lab and environment and cell types).
Staining protocol for surface staining (IL10-R) is similar to that of CD4, CD8 or CD 14.
Hope the article attached is informative for you.
Jitendra Kumar Shandilya thanks for your reply!
I was majorly wondering about the staining protocol, intracellular vs surface stain and if I should consider a specific temperature for the staining, as for example chemokine receptors are often better stained at 37°C.
My actual samples are ex vivo from infections, so I would expect some of the lymphocytes to be positive for IL-10R without specific in vitro stimulation.
Valerie Plajer for any surface staining as far as my knowledge 37 degree C for 20-40 minutes in dark is optimum condition.
For intracellular stainings, it varies depending upon the compatible perm buffer i.e. if you use some methanol based buffers (pospho buffers) 4 degree C for 20-60 minutes is optimum condition (mostly for phospho antibodies) while using triton-X 100 15-20 minutes at 37 degree C is best i got. Some buffers recommend to add antibody during perm step while some buffer recommend to stain with Ab after perm step.
So the staining protocol depends upon the Perm bufffer. If you are buying any commercially available Perm buffer, you are suggested to follow the manufacturer's instructions for optimum conditions.