Does anyone try to induce inflammasome activation with LPS ATP and detect cell death with LDH and obtain discordant results?
In macrophages or PMA-treated human THP-1 cells, LPS-treatment "primes" cells for activation of NLRP3 inflammasome. Priming is a first step, which through activation of NF-KB activity, induces the expression of pro-IL-1b and NLRP3. Treatment of "primed" cells with ATP (a "danger" signal or damage-associated molecular pattern), activates the NLRP3 inflammasome, resulting in processing of pro-IL1b and pro-IL-18 to mature IL-1b and IL-18. Increased production of pro-inflammatory cytokines such as IL-1b and IL-18 results in inflammation.
Inflammasomes can be activated in non-myeloid cells, such as epithelial cells, B and T cells.
Thank you for explanation, i tried to induce NLRP3 inflammasome and i did it with LPS+ATP classical way. However, we expected NLRP3 inflammasome activation causes pyroptotic cell death and we would have detected this type of cell death by using LDH release. I am just curious about cell death detection. Have you ever detect pyroptosis in any cell line for 4 hrs LPS priming followed by 1 hr ATP treatment?
Hi Kamer,
ATP should actually kill cells after 4 hours in this scenario. Just something to keep in mind.
Regarding your experiment, you need to be certain that your ATP is pH balanced to 7.4 before using it in these experiments. If you use the ATP solution directly it will be too low pH. Very low or very high pH kills the LDH reaction.
If you want a secondary readout, I would recommend doing your experiments in the presence of propidium iodide. Unlike other dyes (such as TOPRO3), PI does not pass through ATP-activated P2X7 channels and get into the nucleus and will only stain dead cells.
i will try to increase pH of ATP solution which i will prepare. And i will contact you. Thanks
Actually, yes i checked wb casp1 and more, i just want to be clear about cell death of lps induced inflammasome.
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