Hello everyone.
I'm performing CD4+Foxp3- (Tcons) and CD4+Foxp3+ (Tregs) cocultures. Where I have titrated the Tregs with same counts of Tcons in several wells to see a suppressive effect of Tregs.
The cells are cocultured for 72 hours after which they are harvested, stained with a live/dead dye, stained for surface markers and then fixated and stained for internal markers like Foxp3 and Ki67. I used the dilution for Ki67 as 1:1000 and the staining was done for 30 minutes in the fridge. I also had an FMO for this experiment.
My concern is that, I could not see any signal in the Ki67 channel.
However, I noticed descent live population an quite high total number of events per sample. Therefore, I expect that the proliferation has taken place and I should see Ki67 expression in my samples.
Please suggest if I need to change any settings in my staining system or any improvisation that I can bring to my experiment.
Thanks and regards
Dear Gayatri Tandon
For some considerations concerning improved Ki67 staining in T cells, please check my earlier answer to a similar question
https://www.researchgate.net/post/Problems-with-Ki67-staining-to-look-at-T-cell-activation-proliferation
In brief, I would try using CFSE to see, if your staining problem persists.
In addition, you may consider the following publication from the Buckner lab for human cells.
https://www.sciencedirect.com/science/article/abs/pii/S0022175917301850?via%3Dihub
For murine cells, check the methods section of
Article CTLA-4 control over Foxp3+ regulatory T cell function
All the best & good luck with your experiment,
Michael
