I have been trying to establish an ATP release assay in cultured iPSC-dopaminergic neurons. I put 225 uL of a buffer (128 mM NaCl, 1.9 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 10 mM d-glucose, 10 mM Hepes-NaOH (pH 7.4), 2.4 mM CaCl2, and 0.2% (wt/vol) BSA; same recipe as used in Kato et al PNAS 2017 to measure ATP release from cultured cells) per well of a 24 well plate containing 500,000 iPS-dopaminergic neurons per well. I incubate for 30 min at 37C, and then remove 75 uL of supernatant for my baseline ATP measurement. I then add 75 uL of the same buffer containing 120 mM KCl so that the final concentration of KCl in the well is 40 mM. I incubate for 5 min at 37C and collect the supernatant. I place the supernatants on ice, spin at 4C (2 min at 1000g) to pellet debris, and run the samples in triplicate using the luciferin-luciferase assay to measure ATP (Invitrogen kit). I was able to evoke ATP release from day 75 dopaminergic neurons using this method, but this worked only once. Multiple repeats using the same buffer and d70+ dopaminergic neurons (which are electrically active on calcium imaging experiments) have all failed: the sample ATP values are near blank (blank is just the buffer I use). I get a good standard curve each time so I don’t think it is a kit issue.

I wanted to ask:

Please let me know if you have any suggestions for how I can troubleshoot this assay.

Many thanks,

Aishwarya