Hi Ankitha.

I assume that you use anti-CD3 in your TH17 differentiation culture? What concentration / clone do you use? Do you coat the plate with the anti CD3? Or do you use soluble?

In our hands using irradiated (T cell-depleted) splenic feeders together with soluble anti CD3 2.5ug/mL final (clone 145-2C11) gives better amounts of IL-17+RORgt+ cells (>50%) and better viability (TGFb 5ng/mL, IL-6 20 ng/mL, IL-1b 20 ng/mL and 10ug/ml of of anti-IFNg and anti-IL4). We also get a proportion of RORgt+ cells that are IL-17-, I don't think it is anything unusual.

Hope this helps!

I have had long experience with this and other lineages, although there are several levels of detail (most recently reported in http://elifesciences.org/content/4/e05920). RORgt is not a good marker. I would stain at the same time for Foxp3; your TGFb concentration seems on the high side and may be dominating.

I forgot to say that to my knowledge there is no RORgt-specific antibody commercially available. The available antibodies stain for both RORg1 and RORg2 (RORgt) which only differ by one amino acid. That could be why we detect RORg1/g2+ cells that are IL-17-.

Thanks for the help, Bernard and Aymeric :)

I use 10ug/ml anti-CD3 coated plates and soluble anti-CD28 (2ug/ml).

It helped to know that RORgammat is not a great marker since IL17 -ve cells also express them :)

I will try focussing on IL-17 staining. But somehow I think I am either not being able to achieve Th17 polarization or maybe my [Golgi-plug inhibition + Intracellular staining] is not working well. In case the latter is true, I wanted to make sure I have an alternate non-cytokine Th17 marker that can be detected and compared. 

Could you suggest some?

Also, how many days of polarization should be maintained to achieve maximum IL-17 expression? I harvest the cells on day 5.

Is splitting on day 3 advisable? I add half the concentration of cytokines on day 3.

On day 5, I pool the 96 well triplicates in a 24 plate well and then add PMA/Ionomycin and GolgiStop. Some reports suggest that gologi stop should be added 2hrs after PMA/Ionomycin treatment to achieve greater IL17 expression. I usually add all 3 together. Does that significantly affect the expression?

Thanks.

Dear Ankitha,

I am performing Th17 in vitro cultures from BL6 mice and I started with a few percents like you, but after a lot of slights improvements, I reached 60% IL17A+ cells at day 3 or day 5, so there is still hope  !

For CD3/CD28 stimulation I would suggest using non-treated surface plates (not with the nunc covering because antibodies fix less to the plastic then) - do you manage to make all your cells proliferate ? if no, untreated plates might help (and on top of that Th17 like strong TCR stimulation while they are inhibited if CD28 is too high - I use 1 ug/mL anti CD3 and CD28, both coated, on untreated plates)

For the medium, IMDM is much better than RPMI for Th17 (for whatever reason !)

Maybe you can try one well where you keep the cells with TCR stimulation for 7 days (but adding media regularly), it is less physiological but you get more IL17A+ cells -

You can add 10 or 20 ng/mL IL23 each time you add fresh medium, it helps maintaining IL17 for long term,

I use 10 ug/mL anti IFNg instead of anti IL4, maybe it can help

For rorgt, it is normal that you don't see much of it because you look too late I think, in my cultures, RORgt is maximally stained at 48 hours and gets down to very low levels at day 5 or 7, but these cells are still highly producing IL17,

Note that Th17 cells don't like to be crowded (the more cells the less Th17), so I would suggest 500 000 cells/mL or less as a start,

Maybe you can lower the dose of TGFb to 1 ng/mL or less,

For PMA-Iono, there is two schools, some people prefer to wait before putting Brefeldin or golgi stop to have less background (to be sure what was being synthetized is not kept but only what you induce by restimulation), but I don't like the idea that cells can influence each other during the PMA/iono restimulation, so I put brefeldin A since the beginning. I didn't see much difference between the two methods regarding IL17+ cells, 

I use the same antibodies as you mentionned,

Feel free to contact me by mail if you want to discuss it more in details, 

Best Regards,

Philippe

Apart from the most popular cytokine pack to drive Naive Th cells to Th17 phenotype, you may also need to look at the age of mice sacrificed, we usually got decent IL17 readout with mice of 6 - 8 weeks old.

1 ug of anti cd3 works best. over activation doesn't work so well. 

I forgot to mention that it is also better to transfer the culture to a new 96 well plate after 3 days or so (and split it in half). The viability will be much better. In my ends coated anti-CD3 is harmful for the cells beyond 3 days and actually kills them.

Thank you so much everyone for all the inputs!!!!!

Troubleshooting is much easier now... :)!!!!