A solution of ATP is mildly acidic in nature. As acidic pH is known to have a stimulatory role in inflammaosme activation, i was wondering if one has to maintain the pH at 7 or just leave it as it is?
Hi Atul,
I prepare first a 500 mM ATP stock in RPMI+PenStrep+L-glut (pH indicator in the medium will turn yellow due to acidity) and then add 5 M NaOH until the media color just and just shifts to slightly reddish to neutralize the solution. Then I calculate the new ATP stock concentration according to added NaOH volume. If you do the titration once carefully with small NaOH volumes, you will easily find a standard volume ratio to add to your stock solution.
While acidic pH alone will stimulate the NLRP3 ifnlammasome, it also strongly blocks ATP-induced P2X7 receptor signaling required for ATP-induced inflammasome activation, so the overall effects of acidity might be rather unpredictable.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2697781/
It's generally recommended to pH balance your ATP before using it. You can use litmus paper if you want to avoid contaminating your stock. I've found that making your ATP in the buffered medium that you use for your experiment helps reduce drastic fluctuations.
Thanks Jordan for your answer
To be clear, do you make the stock in the buffered medium? I use RPMI-1640 as my medium. So do i make the stock in complete RPMI or just RPMI (without serum and all)
Appreciate the help
Atul
It's been some time since I used ATP for NLRP3 stimulation, but I would generally prepare a 100 mM stock in PBS and dilute that into complete medium for something like an LDH assay. If, on the other hand, I was collecting supernatants to concentrate for a WB then I would do the ATP stimulation in serum-free medium.
Hi Atul,
I prepare first a 500 mM ATP stock in RPMI+PenStrep+L-glut (pH indicator in the medium will turn yellow due to acidity) and then add 5 M NaOH until the media color just and just shifts to slightly reddish to neutralize the solution. Then I calculate the new ATP stock concentration according to added NaOH volume. If you do the titration once carefully with small NaOH volumes, you will easily find a standard volume ratio to add to your stock solution.
While acidic pH alone will stimulate the NLRP3 ifnlammasome, it also strongly blocks ATP-induced P2X7 receptor signaling required for ATP-induced inflammasome activation, so the overall effects of acidity might be rather unpredictable.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2697781/
Thanks for your suggestions Kristiina !
Will setup the experiments and see where it takes me
Thanks,
Atul
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