Atp fluorometric assay kit

H9C2 cells were seeded in black 96-wells at 5 × 10³ cells/well and treated with an unrelated His-tagged protein, CTRP7 or CTRP9 (4 µg/mL) for 24 h, unless otherwise stated. Cellular ATP content was measured by phosphorylating glycerol, resulting in a fluorometric product proportional to the amount of ATP, with the ATP fluorometric Assay Kit (Sigma) at 535 nm (excitation) and 587 nm (emission) on an Infinite^® M200 microplate reader (Tecan, Männedorf, Switzerland). In order to estimate cell viability, cells were loaded with the CellTiter-Blue^® Reagent (Promega, Walldorf, Germany), according to the manufacturer’s instructions, and incubated for another two hours. Afterward, fluorescence was recorded at 560 nm (excitation) and 590 nm (emission). For the measurement of cellular reactive oxygen species (ROS) production, cells were loaded with 10 µM 2′,7′ –dichlorofluorescin diacetate (DCFDA, Sigma) for 30 min, washed with PBS and, afterward, incubated for 24 h with CTRP7 or CTRP9. Fluorescence was recorded at 485 nm (excitation) and 535 nm (emission). All data are expressed in relation to the according controls.

Chlamydomonas cells containing 50 μg chlorophyll were pelleted from cultures and resuspended in 925 μl of 0.5 M tris-HCl buffer (pH 7.5). A total of 75 μl of 70% perchloric acid (Sigma-Aldrich) was added to the cell samples, which were then vortexed for 15 s to both lyse the cells and deproteinize the lysate. The samples were then incubated on ice for 1 min, neutralized with 4 M KOH, mixed thoroughly by vortexing, incubated for an additional minute on ice, and then centrifuged for 1 min (3200g). A total of 500 μl of supernatant was transferred to a prechilled tube that was then flash-frozen in liquid N₂ and stored at −80°C. A total of 25 μl of the supernatant was used for ATP quantification based on the “ATP Fluorometric Assay Kit” from Sigma-Aldrich (MAK190). Fluorescence was quantified in a Tecan Safire plate reader. For measuring the ATP/ADP ratio, we diluted the same samples (1:250) and used the “ADP/ATP Ratio Assay kit” (Sigma-Aldrich, MAK135). Bioluminescence was determined using the TD-20/20 luminometer from Turner Designs.

Cellular ATP levels in FACS-sorted RPMs (10000 cells/sample) were determined using ATP Fluorometric Assay Kit (Sigma-Aldrich, MAK190), as per the manufacturer’s instructions.