I am doing FM1-43FX staining with the addition of ATP and nonselective antagonist of P2x, 200 uM Suramin. I get robust inhibition of dye uptake in the presence of Suramin. However I get positive staining in the presence and absence of ATP. I want a protocol that would allow a reduction of ATP levels in my cell culture so that when I add ATP I see a robust response.
You might have to raise the pH of the media upto 9.0 for the phosphatase to work effectievly. I am not sure if P2X receptors potentiate with extracellular pH changes. Apyrase is a better option as it works slightly below the physiological pH. But if you ask me there is no need to mop up the ATP before addition. If you are getting a decent increment in the stain after ATP addition, despite the background, you might as well go with it becasue it is much closer to the natural physiological response. As long as you have an image before ATP addition showing the background stain, any good journal will accept your data. You can also quantitate the increment in staining by ImageJ and add a bar chart indicating the increase with error bars and statistical significance.
Hallo! May be this article will you useful
https://www.pnas.org/content/pnas/early/2018/05/30/1717724115.full.pdf
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