Hi Cybel,

In our lab we differentiate BMDM until day 6, on which we seed them, and work with them the next day (day 7).

I generally prime my cells with 500 ng/ml LPS or Pam3 for 2 hours and then I stimulate with TcdA or TcdB (1 µg/ml) for 5 hours.

What's the source of your TcdB? I have had contamination in my prep when I purchased it from Enzo Life Sciences but the one from ListLab was clean. I have had no problems though with TcdA, and use it on a regular basis. IL-1b, casp1 and LDH signal are specific, as it is completely flat in Pyrin KO BMDMs.

Try going up with your toxin to 1 µg/ml. If you think it is too much do it at 500 ng/ml for 5 hours or overnight. If overnight, I would wash away the LPS to avoid the well known IL-10 mediated tolerance.

Hope this helps.

Dear Pedro,

Thank you so much for your valuable comment and advice.

In all my experiments, TcdB from R&D systems was used but based on the advice of a collaborator, TcdB might have been the issue,

I ordered TcdB from ListLab as suggested by both of you and will proceed with the experiment as soon as my BMDMs are ready.

I will keep you posted.

Thank you again:)

Best

Cybel

Dear Pedro,

I just wanted to inform you that my experiment finally worked:

I actually switched to the TcdB from ListLab and got a robust activation of pyrin in primed BMDMs stimulated with TcdB (500 ng/mL, 5h).

Thank you again for your advice,

Best

Cybel

Hi Cybel,

Glad it worked out for you!

Ideally, it would be better to express and purify these toxins in-house since they cost a lot of money, although more convenient if there is no protein facility around!

Good luck on your research.

Best,

Pedro