Xf real time atp rate assay kit

The respiration activity in primary hepatocytes was measured at 37 °C by high-resolution respirometry with the Seahorse Bioscience XF24-3 Extracellular Flux Analyzer. Succinate (10 mM) and rotenone (2 μM) were used as substrates to quantify State 2. State 3 was initiated with ADP, State 4 induced with the addition of oligomycin (State 4o), and FCCP-induced maximal uncoupler-stimulated respiration (State 3u) were sequentially measured. The normalized data are expressed as pmol of O₂ per minute or milli-pH units (mpH) per minute, per µg total protein. The dual-ATP production rate was assessed using Seahorse XFe24 Analyzer (Agilent Technologies, Santa Clara, CA, USA); simultaneous reads of ATP production from glycolysis and mitochondria were performed using a label-free technology XF Real-Time ATP Rate Assay kit, as described in the User Guide (Agilent Technologies).

Basal OCR, extracellular acidification rate (ECAR), and ATP production rate were measured using XF Cell Mito Stress Test kit, XF Glycolytic Rate Assay kit, and XF Real-Time ATP Rate Assay kit on Seahorse XF Analyzer (Agilent Technologies). The sensor cartridges used for measuring the oxygen flux was equilibrated in an XF Calibrant (Agilent Technologies) for 16–24 h before the experiment in a 0%-CO₂ 37 °C incubator. The granulation tissue from the wounds taken from mice after 8 days of wounding was carefully dissected and rinsed with unbuffered Krebs-Henseleit buffer (KHB) media. The tissue was placed at the bottom of the XF24 Islet Capture Microplate (Agilent technologies) and covered with a mesh. Four hundred and fifty microliters KHB medium was added to each well containing the tissue and equilibrated in a 0%-CO₂ incubator for 30 min. The cartridge was then placed on the assay plate and run in the XF analyzer using an optimized protocol to measure basal OCR. For analysis in cells, HDFs were transfected with negative control mimic or miR-210 mimic and treated with normal (5.5 mM) or high (30 mM) glucose levels in normoxia or hypoxia. The cells from each condition were then seeded onto an XF24 or XFe96 Cell Culture Microplate and (Agilent Technologies). The results were normalized to protein concentration or cell number as indicated.

For ATP production rate assay an Agilent Seahorse XF Real-Time ATP Rate Assay Kit was used and HEK293 MUL1(−/−) and HEK293 MUL1(+/+) cells were plated as described above and ATP measurements were recorded followed by Oligomycin injection in port A (56 µl) at 1.5 μM final concentration, and Rotenone/Antimycin injection in port B (62 μl) at 0.5 μM final concentration. Data analysis was performed using Report Generators software for Real-Time ATP Rate Assay.