Make sure TGF-beta is acidified before adding to the cells for differentiation of Tregs.

Sharvan Sehrawat Yes, the TGF-beta has been acidified. It was reconstituted at 100 μg/mL in sterile 4 mM HCl containing at least 0.1% BSA.

Dear Weiwei Wang,

Some ideas, if it helps:

- Which time-point do you use? In vitro, Foxp3 is not necessarily stable, I would suggest to look at day 3 and day 5, it should be maximal at day 5

- Normally I stop TCR stimulation at day 3 and replate the cells on a new plate with fresh medium. Continuous TCR stimulation after day 3 tends to kill Foxp3+ cells

- Normally we use IL2 for the iTreg culture, it helps to survive. The dose depends on protocols, I used 20ng/mL

- maybe checking the efficiency of activation, for instance by labelling with CTV or CFSE low dose before activation. It might be that the cells are not stimulated enough and then you also get low Fop3 induction. Suggestion: if TCR stimulation is too low,

- I used this clone for anti CD3 (2C11) and did both coated on the plate. As a trick, I do coating 3 hours in PBS 4 degrees, but we do not use the normal cell culture plate. Instead we use 'untreated plates' i.e. non-NUNC-treated. The antibodies stick better on the plate. I use 1 ug/mL for both anti CD3 and anti CD28. It makes sense you use higher concentration if soluble.

- Normally we sort 'clean' naive Foxp3- cells, either using huCD2 reporter mice, or using extracellular markers, like CD44, CD62L, CD25 or even GITR. Depends on the available sorting. I would say in theory it should work good as well on bulk CD4+ cells but maybe setting up the protocol with a clean population helps.

- (independent suggestion) Maybe you can have a 'Th0' control without TGFb to see if your serum already contains TGFb. We have seen that depending on the FCS you cal already get some Foxp3+ cells without TGFb.

I am happy to get into details by message and share protocols.

Philippe August Robert thank you very much, i will make a try following your advance.