Invitrogen atp determination kit

P. aeruginosa polyP levels following nutrient deprivation in morpholinepropanesulfonic acid (MOPS) minimal medium were assessed as described previously (8 (link)), with slight variations. Briefly, overnight cultures were grown in 5 ml LB in the presence of inhibitor or DMSO as indicated. Cultures were centrifuged for 10 min at 3,900 rpm to pellet cells, which were then resuspended in MOPS minimal medium containing 100 μM phosphate and the indicated quantity of inhibitor or DMSO. Cultures were incubated at 37°C for 2 h to allow polyP accumulation and then subsequently pelleted, resuspended in guanidinium thiocyanate (GITC) lysis buffer, and boiled at 95°C for 5 min. PolyP was purified via silica spin column as described previously (8 (link)), with the exception of using 50 nM PPK2A instead of E. coli PPK to convert polyP to ATP. An Invitrogen ATP determination kit (ThermoFisher) was used per the manufacturer’s instructions for ATP quantification. Luminescence was recorded on a SpectraMax iD3 microplate reader (Molecular Devices).

2 ml tubes were preloaded with 250 μl of buffer BI (3 M HClO₂, 77 mM EDTA). 1 ml culture sample was added, vortexed and incubated (lysis, 15 min on ice). 600 μl of BII (1 M KOH, 0.5 M KCl, 0.5 M Tris) were added (neutralization). Samples vortexed and incubated (10 min, on ice), centrifuged (10 min, 0°C, 12 000 g), flash-frozen in liquid nitrogen and stored at –80°C. Extracts were thawed on ice and centrifuged (10 min, 0°C, 12 000 g). 200 μl samples were added either to 320 μl of BIII/PEP (100 mM HEPES, 50 mM MgSO₄·7H₂O, adjusted to pH 7.4 with NaOH, and 1.6 mM phosphoenolpyruvate (Sigma-Aldrich, 860077)) for ATP quantification or BIII/PEP + PK (BIII/PEP with 2 U/μl pyruvate kinase, (Sigma-Aldrich, P1506)) for ATP + ADP quantification, incubated (30 min, 37°C), and heat-inactivated (10 min, 90°C). ATP concentrations were determined using the Invitrogen ATP determination kit was used (ThermoFisher: A22066). 10 μl of each PEP or PEP + PK-treated sample was loaded in a white 96-well plate with solid bottom and kept on ice until the reaction was started. The luciferase master mix was scaled down in volume, and 90 μl of master mix was added to each well. Luminescence was recorded using a BMG Clariostar. ATP concentrations were calculated from a standard curve on the same plate.