Hi all,
Our lab is trying to activate pyrin inflammasome in bone marrow-derived macrophages with TcdB toxin from Clostridium difficile. We are not a microbiology lab and we have to purchase the TcdB toxin from R&D (catalog# 6246—GT-020). I am wondering if anyone used this toxin and successfully activate pyrin inflammasome in BMDMs?
This toxin came in as liquid at a concentration of 0.575mg/ml, and I tried 0.1, 0.2, and 5 ug/ml according to the publication. Unfortunately, I didn't see any pyroptotic cells under the microscope as long as 24 hours.
Briefly, 1x10^6 WT BMDMs were seeded in the 6-well plate, and BMDMs were primed with 500ng/ml LPS for 4 hours. The cells were then washed, and the medium was replaced with FBS-free medium. TcdB toxin from R&D (6246-GT-020) was added directly to the wells.
Are we missing any critical points here?
Thank you in advance!
Best.
Hope you find an answer as I am struggling with the same problem... TcdB resusp in 500mL water does not trigger cell death or IL-1 in my THP1 monocytes, 10ul toxin in 400ul cells with 500,000 cells for 2 to 24 hours with LPS priming
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