ATP in water is highly acidic, and rapidly turns the phenol red in my medium yellow when I add it for P2X7 stimulation. A recent book chapter in "The Inflammasomes" (Springer Protocols) suggested preparing 500 mM ATP in solution with 500 mM NaOH. I measured the pH of this and it was quite basic (if I remember correctly).
I've recently been preparing stocks of 100 mM ATP in 800 uL water and adjusted to approximately pH 7 with 200 uL of 1 M NaOH then freezing aliquots at -80C for subsequent use.
Since the literature rarely says anything other than "5 mM ATP" for P2X7 stimulation and does not describe the composition of the literature, I was wondering what other researchers are using for their preparation.
Hello, P2X receptors are very sensitive to the pH of the solution, depending of the subunit could increase the current or decrease it. In this way, could be very important maintain pH close to physiological to determinate in its real proportion the participation of activation of P2X receptors.
Thank you for the answer, Francisco. I wonder, then, why it's almost never mentioned in the methods of the literature. The preparation of an ATP solution is almost always left out.
Hi Jordan,
I also prepare 100mM solutions of ATP and adjust the pH to 7 (approximatlely). It works fine.
Hi Jordan,
For working on P2X7 receptors, we routinely prepare ATP in distilled water or in sodium chloride buffer at 100mM and pH to 7.4 with 5M NaOH. Stocks can be kept frozen at -80 degrees for several months with no deterioration. We don't re-use thawed ATP.
Information regarding preparation of ATP should be included in the methods section and I will certainly keep this in mind in future for my articles.
Hope this helps you,
Leanne
Hi Jordan,
I was wondering about the very same thing some time ago, and I decided to test the IL-1beta response of LPS-primed human macrophages to 5 mM ATP by using both a neutralized and an unneutralized ATP stock solution. Although it has been reported that the P2X7 receptor responsible for the ATP-induced inflammasome activation and IL-1beta secretion is strongly inhibited by acidic pH, the IL-1beta responses were similar. This may be related to the fact that the macrophages seem to regulate their surrounding pH back close to physiological quite rapidly (in 10min marked recovery of pH as judged by media color; after 30 min medium pH around 7). Of course the 5 mM ATP concentration is also very high and may thus overcome the inhibition caused by mild acidosis.
Good luck with your studies,
Kristiina
Thank you for your helpful responses Leanne and Kristiina. I also noticed that the medium quickly rebounds to normal or close to normal pH, but I had some fear of damage caused to the cells by the high pH (particularly in the case of an LDH assay).
I've recently been doing very much the same as you Leanne and have been having fine results. It's good to know that other researchers agree before I get too deep.
Cheers!
We have also had issues with pH following ATP but also with other types of compounds to test for inflammasome activation. I am wondering if Kristilna Rajamaki examined the IL-1beta response by ELISA or by Western Blot.
I need to look for the reference to confirm, but I remember to had read that its important to ATP be with 4 negative charges to activate P2X7. So, we normally adjust the pH of ATP solution with TRIS, to pH ~6 - 7.
Hi G. Jean Harry,
I wonder as well. I will say, however, that I found the best results when my ATP was dissolved in pH balanced culture medium, and did not need to neutralize the ATP. Western blot for caspase-1 and IL1b, as well as ELISA for IL1b, speck formation by IF and YVAD-FLICA all ended up as expected. See the data in the paper linked below for more.
Article K+ regulates Ca2+ to drive inflammasome signaling: Dynamic v...
Thanks Jordan
getting the mature IL-1beta is not as easy as originally thought. This paper is very helpful
Hi Jean,
We analysed the IL-1beta response by ELISA in this case (from primary human monocyte-derived macrophages). I've been routinely neutralizing my ATP stock solutions for quite some time now; similarly, I've neutralized certain inhibitor compounds (both basic & acidic ones) that tended to affect cell culture media pH. Quite naturally I started paying more attention to pH changes after finding out that acidic pH triggers NLRP3 inflammasome activation and basic pH tends to reduce it.
Dear Kristiina,
P2X7R is activated by extracellular ATP in its tetra-anionic form. Thus, pH and concentration of divalent ions such as Mg2 are important factors that can decrease the real concentration of ATP. ATP possesses several groups with different acid dissociation
groups. At neutral pH, ATP is in a tetra-anionic form (and more stable than at extreme pH).
I think that it's a very good idea to work at pH around 7 and in buffers containing low concentrations of Mg2.
Very best wishes.
Jean K.
Thanks
we are a bit concerned with examining potential modifiers of the inflammasome activation and if those modifiers might nonspecifically alter the response due to pH. One thing we did find was the the IL-1beta ELISA was not a reliable readout for mature IL-1beta as it detects both pro and mature forms and while we find IL-1b released into the medium with ELISA we also find that this is primarily the pro form and not the mature protein. This may end up being a cell type differences but it has led us to always run a western even though it takes a large amount of cells.
Jean K. : Thank you for the very detailed description of the underlying chemistry.
Jean H. : Which ELISA were you using to detect mature human (?) IL-1beta? We have been concerned on the cross-reactivity with the proform, but for the R&D Duoset human IL-1b ELISA we're using, we have been getting pro/mature corresponding nicely to WB. Similar to your results, we detect much more proform in media (10-fold), we even checked that with R&D human pro-IL-1b ELISA.
@ G. Jean Harry Hi, I'm fresh to make some basic researches in the inflammatory field and i'm confused about the words that you find IL-1b released into the medium with ELISA and you also find that this is primarily the pro form and not the mature protein. You meant the Pro-1B form can also be secreted into the supernatant medium? and i also wanna know the IL-1B antibody you use could detect both of pro- and cleaved IL1B so that you can make this conclusion?Hope to receive your reply.
Best wishes
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