Atp fluorometric assay kit

ATP concentrations were measured in cells using the Fluorometric ATP Assay Kit (#ab83355) from Abcam, Cambridge, United Kingdom. Briefly, following the experiment, cells were harvested, counted, and protein-precipitated according to manufacturer instructions. After adjusting the samples' pH to between 6.5 and 8, samples were incubated with the kit reagents according to the fluorometric protocol, and ATP concentrations were normalized to 1 × 10⁶ cells.

Tissue ATP content was measured in frozen fetal heart ventricles of each treatment group. Tissue lysates were generated by homogenizing tissue, sitting on ice, in 100 μl ATP Assay buffer with a Dounce homogenizer. ATP content was obtained using a Fluorometric ATP Assay kit (Abcam, Cambridge MA) by following the manufacturer’s protocol. Briefly, 50 μl of Reaction Mix from the assay kit was mixed with 50 μl of lysates in a 96-well microplate. Fluorescence of signal was detected at excitation at 535 nm and emission at 587 nm on a microplate reader at 5-min interval for 30 min (BioTek, Winooski, VT). Sample ATP levels were measured in duplicate and averaged. ATP content of each sample was derived from the standard curve, normalized to their mg protein, and calculated as nM/mg protein.

Fresh tissue was isolated from posterior left ventricular wall, weighted and placed into a 2N perchloric acid solution, homogenized and then incubated on ice for 30 min. Samples were then centrifuged (13,000 g for 2 min at 4°C) and 100 μL of supernatant was added to 500 μL of assay buffer. Samples were then neutralized with 2M KOH, vortexed and centrifuged (13,000 g for 15 min at 4°C) to remove PCA. The resulting supernatant was then used in the Fluorometric ATP Assay Kit (Abcam) according to the manufacturer’s instructions.

Mammary and liver total ATP content was measured using Fluorometric ATP Assay Kit (ab83355, Abcam). Briefly, approximately 10 mg of tissue was washed in cold PBS, and then homogenized in 100 μl ice cold 2N perchloric acid (PCA) with a Dounce homogenizer (Thomas Scientific) using 10–15 passes. Samples were incubated on ice for 30–45 min, and then centrifuged at 13,000 g for 2 min at 4°C. Supernatant was transferred to a fresh tube, and volume was brought to 500 μl by adding ATP assay buffer.
PCA was precipitated by adding 100 µl of ice-cold 2 M KOH and sample was vortexed A 5 μl aliquot was used for testing using pH paper, and pH was adjusted to 6.5-8. pH by addition of 0.1 M KOH or PCA. Samples were centrifuge at 13,000 g for 15 min at 4°C and supernatant was collected and measured fluorometrically at Ex/Em=535/587 nm (Spark 10 M, and Plate: Thermo Fisher Scientific-Nunclon 96 Flat Black with transparent bottom, NUN96fb). The concentration was calculated as described in manufacturer's protocol and expressed as ng/µl.

Cells grown in 12-well plates were treated with 100 μM MPP⁺ or 4'I-MPP⁺ in KRB-HEPES for 6 h at 37 °C. After the treatments, cells were suspended in 1 mL of ice-cold KRB-HEPES and 50 μL samples were withdrawn for protein quantification. The remaining cell suspensions were centrifuged for 3 min at 3,500 x g at 4 °C and the cell pellets were treated with 1 mL of lysis buffer (0.1 M Tris, 1% Triton X-100, pH 7.5) for 2 min and centrifuged at 3,500 x g for 3 min to remove the cell debris. The ATP contents of the supernatants were measured using a fluorometric ATP assay kit (BioVision, Milpitas CA, USA) following the manufacture’s guidelines_. All ATP levels were normalized to the protein content of each sample.