Atp assay kit

The ATP levels were measured using an ATP Assay Kit (BioVision, CA, USA), following manufacturer’s protocol. The absorbance of samples and standards was measured with a BioTek ELz800 microplate reader (BioTek, VT, USA). Intracellular ATP levels were determined based on a standard curve and calculated as nmol/µL (per 10⁵ cells) and expressed as a relative percentage of that of the control.

Fructokinase activity in HK-2 lysates was determined by measuring the amount of fructose-dependent ATP depletion in a kinetic reaction as follow. As control, HK-2 cells deficient for KHK expression were employed to calculate background ATP depletion during the length of the assay. The ATP depletion cell lysate reaction contained 100 μg of lysate protein, 50 mM imidazole, 4 mM magnesium chloride, 1 M potassium acetate (pH 7.5), 20 mM n-acetylglucosamine, 40 mM sodium fluoride, 5 mM fructose and 5 mM ATP in a total reaction volume of 200 μl. Lysates were preincubated with different concentrations of luteolin in the reaction mixture for 15 min at 37 °C. After adding in the fructose (except for negative controls), the reaction was continuously shaken and incubated at 37 °C for 120 min. Samples of each reaction were taken after 2 min and 120 min for measuring ATP levels. ATP levels were measured using an ATP Assay Kit (BioVision, Inc., Milpitas, CA). Five microlitres of sample was added to 95 ul of the ATP reaction mixture and readings were taken at 570 nm. The change in ATP levels were calculated and the percentage ATP inhibition was determined. IC₅₀s for inhibiting ATP depletion was calculated from the dose response (0.1–500 μM).

Cells were cultured in 6-well plates at a confluence of 1 × 10⁶ cells/well with or without RJ for 24 h and then incubated with or without MPP⁺ for 24 h. Collected cells were lysed, and the cell protein was precipitated and neutralized using a Deproteinizing Sample Preparation Kit (Biovision, Milpitas, CA, USA). Then, a 96-well plate containing 50 μL of sample and 50 μL of the reaction mixture of the ATP assay kit (Biovision) was incubated at RT for 30 min and read at 570 nm. The amount of ATP in the sample was calculated using a standard curve.

To measure the amount of ATP in cell culture supernatants, samples were centrifuged at ice-cold at 14,000 rpm for 2 min and the supernatants were stored at −80 °C prior to analysis. The amount of ATP was measured using ATP Assay Kit (BioVision Inc.; Milpitas, CA) according to the manufacturer’s instructions.

The ATP content was quantified from frozen whole lung tissue using ATP Assay Kit (Biovision, Inc. Milpitas, CA), following manufacturer’s instructions and previously described protocol [64 (link)]. Briefly, frozen lung tissues were pulverized at − 80 °C followed by homogenization using assay buffer provided by manufacturer. Total Protein was quantified using BCA (bicinchoninic acid) assay kit (Thermo Fisher Scientific, Waltham, MA). The standard curve for ATP was generated over a range of 20 nM to 1 nM. The phosphorylated glycerol from lung homogenates was measured at 570 nm using SpectraMax®iD5 (Molecular Devices, CA) plate reader after incubation with the probe for approximately 90 min. All results were normalized to total protein measured using BCA (bicinchoninic acid) assay kit (Thermo Fisher Scientific, Waltham, MA) for quantification.