Atp bioluminescent assay kit

Cellular ATP levels were quantified using ATP Bioluminescent Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s guidelines. Briefly, exponentially growing cells under both SM^nlglu and SM^lowglu conditions in the presence or absence of FLC were washed three times with PBS. The cellular ATP was first isolated with the trichloroacetic acid method (TCA) as previously published [32 (link)]. 10⁷ cells were mixed with 5% TCA and alkaline lysis solution (1% sodium dodecyl sulfate and 0.2 N NaOH) and vortexed for 15 min in the presence of sterile acid-washed glass beads to initiate lysis. After vortexing, the cell lysates without the glass beads were transferred and boiled at 100 °C for 10 min, cooled and diluted 1:50 with ATP assay mix dilution buffer supplied with the ATP Bioluminescent Assay Kit (Sigma-Aldrich, St. Louis, MO, USA). Diluted sample was mixed with 100 µL of ATP assay mix solution supplied with the ATP Bioluminescent Assay Kit in white-opaque bioluminescent 96 well plates (Corning-Costar, Cambridge, MA, USA). The mixture was incubated in the dark for 3 min and then bioluminescence was measured with the spectrophotometer (SpectraMax I3X). ATP standard solution was used in serial dilution to generate a standard curve. This standard curve was used to interpolate the cellular ATP levels. The assay was performed in biological replicate.

ATP content was measured by a commercial bioluminescent assay (ATP bioluminescent assay kit, Merck) according to the manufacturer’s instruction and as previously described [27 (link)]. Briefly, ATP was extracted by boiling the samples in a solution containing 100 mM TRIS, 4 mM EDTA, pH 7.75. After centrifugation at 10,000× g for 60 s, samples were diluted at 1:50 in dilution buffer (FLAA, Merck). To obtain bioluminescence measurements with a standard luminometer, 100 μL of supernatant was mixed with 100 μL of luciferin-luciferase solution. The standard curve of ATP was obtained by serial dilution of 2 μM ATP solution.

The ATP level was measured in cell lysates as previously reported^{41 (link)}. Briefly, cells were detached with trypsin, washed once with HBSS, and resuspended in a buffer containing 0.25 M sucrose, 50 mM HEPES, 0.5 mM EDTA, 4 mM MgSO4, pH 7.4. Aliquots of cells were lysed in DMSO and total cellular ATP was measured using a Luciferin–Luciferase assay kit (ATP bioluminescent assay kit, Merck KGaA, Darmstadt, Germany) according to the manufacturer’s protocol. The amount of ATP measured was normalized to the protein content determined by a modified protocol of the Lowry method as reported^{42 (link)}.