Atp assay kit

ECAR was measured using the Cell Mito Stress Test Kit (Agilent Technologies, 03,015–100). The trypsin-digested OC cells were inoculated in a 96-well Agilent Seahorse XF cell culture microplate (approximately 5000–6000 cells per well). The cells spread evenly throughout the well. ECAR was examined using a Seahorse XFe24 instrument at the Shanghai NanoBioscience Co. Ltd.
Glucose Uptake Assay Kit (Biovision, K676-100), Lactate Assay Kit (Biovision, K667-100), ATP Assay Kit (Promega, FF2000), and NADPH Assay Kit (AAT Bioquest; AAT-15272) were used to examine the products of glycolysis in the OC cells referring to the instructions.
An untargeted metabolomic analysis was performed using mass spectrometry, which involves the following steps: sample pretreatment, metabolite extraction, full scan detection in liquid chromatography-tandem mass spectrometry, data pretreatment, statistical analysis, and differential structure identification. Approximately 1 × 10⁷ cells from each sample were used with six replicates. The cells were fixed with 1-mL mixture (2methanol: 2acetonitrile: 1water). Metabolomic analysis and mass spectrometry were performed by Shanghai Applied Protein Technology.

The glucose uptake rate was determined with the Glucose Assay Kit (BioVision); the ATP levels were detected with the ATP Assay Kit (Promega); and the extracellular lactate production was measured with the Lactate Assay Kit (BioVision). All the operations were performed in accordance with the instruction manual. The calculated values were normalized to the protein concentration. The extracellular acidification rate (ECAR) was examined by the Seahorse XF Glycolysis Stress Test Kit (Agilent Technologies) in accordance with the operating instructions in the form of sequential addition to each well with glucose (10 mM), oligomycin (2 mM), and 2‐deoxyglucose (100 mM). The ECAR values were calculated after a standardized cell count and plotted as an average ± SD.

Cellular ATP levels were measured using an ATP Assay Kit (Promega, WI, USA) according to the manufacturer's instruction as previously reported [21 (link)]. Briefly, the proteins of ECs treated with various types of EPC-EXs were harvested and determined by the BCA assay (Thermo Scientific). In 6-well plates, 200 μl protein supernatant was mixed with 100 μl ATP detection working solution. The luminance (RLU) was measured by a fluorescence microplate reader (Thermo Fisher Scientific, Varioskan Flash, USA). The total ATP levels were expressed as nmol/mg protein. The relative ATP level was calculated according to the following formula: relative ATP level = ATP value/protein concentration.

Frozen lung tissue (25 mg) was powdered on dry ice and homogenized in 0.5 mL of 0.5% trichloroacetic acid. We centrifuged the lysates for 2 min at 4 °C and 8000 rpm to separate cleared supernatant from insoluble cell debris. The sample supernatants were buffered with 10× concentrated Tris-acetate buffer containing 10 µL of 0.002% xylenol blue as pH indicator. We used an ATP assay kit (Enliten, Promega, Madison, WI, USA) to estimate the ATP concentration in the supernatant by measuring in the luminescence channel of a Cytation 5 plate reader (BioTek Instruments, Inc., Winooski, VT, USA). The results are expressed in nanomolar ATP per 25 milligram of lung tissue. Tissue lysate extracted from the powdered lung tissue was also analysed using a myeloperoxidase MPO activity assay (OxiSelect™ Myeloperoxidase Chlorination Activity Assay, Cell Biolabs, San Diego, CA, USA) and according to manufacturer’s instructions. The results are expressed in microUnit per milliliter of lung tissue lysate. We used an ELISA-based carbonyl assay (OxiSelect™ Protein Carbonyl Elisa kit, Cell Biolabs, San Diego, CA, USA) to determine the protein accumulation of carbonyl modification in powdered lung tissue lysate according to the manufacturer’s instructions. The results are expressed in nM/mL of lung tissue lysate.

To measure the ATP content in the mitochondrial extract extracted from brain tissue, the extract was centrifuged at 13,000× g for 10 min, and the pellet was mixed with 1% TCA and reacted on ice for 10 min. After mixing with 25 mM tris-acetate buffer (pH 7.7) at 10,000× g for 15 min, the supernatant was used to measure the ATP content, which was performed using an ATP assay kit (Promega Corp., Madison, WI, USA) using a luminometer (GloMax-Multi Detection System, Promega Corp., Madison, WI, USA).

In all, 10,000 cells were plated in each well in a 96-well plate and exposed to VLX600 for 24 or 48 h; ATP was measured by ATP assay kit from Promega.