Dear Fataneh Tavasolian

I briefly wanted to reach out concerning your question.

Please consider the following publication for all things Treg cell isolation and culture.

In brief, they did use Ki67 for assessment of Treg proliferation as CFSE stainings can be finicky due to the fact of spillover into other channels and the fact that it is toxic for cells. 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

For a tried and tested protocol for CFSE staining, you may use the attached protocol. For the results, analysis and FACS gating, please consider:

Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...

Article Ubiquitin Specific Protease 21 Is Dispensable for Normal Dev...

For your experimental design, use the following papers as reference:

https://ashpublications.org/blood/article/113/9/1977/107987/Activated-T-cells-recruit-exosomes-secreted-by

https://ashpublications.org/blood/article/118/22/5851/29204/LFA-1-blockade-induces-effector-and-regulatory-T

In brief, I would use the exosome co-culture model in reference 1 and set-up the experiment the same way, using either blocking antibodies to LFA-1 and/or KO cells from LFA-1 KO mice as negative controls.

I have also attached a general review for LFA-1 signaling in T cells that may help your functional downstream analysis even though that will to some extend depend on the proteins/ RNAs you want to assess by exosome transfer.

https://www.jimmunol.org/content/199/4/1213

All the best & good luck with your experiment,

Michael

Hi,

Make sure to label your CD4 T cells prior to activation while they are all relatively uniform in size, this will help get the best CFSE peaks. You can determine how many times the Tregs have divided by using your strategy. You could also include staining for a marker for active proliferation such as Ki-67, though if your assay isn't very long (3-4 days post-stimulation) it might be high on all of your cells. Good luck!

Just to add up,

You can also consider using Cell-Trace Violet instead of CFSE for convenience in terms of FACS colors, since CTV uses different colors than CFSE (violet, which leaves more colors free for other markers), and it works good as well (CFSE can be tricky to compensate if you use PE for instance).

One thing to consider, CTV or CFSE labelling will kill quite a significant amount of cells (25 to 50% in my case), so it is worth it to calculate the cell number after labelling so that you know how many cells remain to be cultured, and to check they survived the labelling. I stop the labelling by adding medium with high Fecal Calf Serum content before washing, it helps the cells feel better.

As Jim said, it is usually recommended to label the cells at the naive state before activation because they have the same size. If you label activated cells, some are big, some are small, and it gets very difficult to observe proliferation peaks.

Suggestions for controls:

- put aside some cells without labelling to know where is the negative (non-labelled)

- put aside some cells just after labelling but not activated to know where is the '0 division' peak

(these controls you could also record them in the FACS at day 0, or let the cells be in a well without activation).

Also, be careful that after 5-7 divisions the label will be quite diluted, which would suggest to analyze at early time-points. For me, I see the best proliferation peaks between day 3 to day 6 post activation, but avoid only looking at day 10, you might not see anything anymore!

Good luck!