Cell mito stress test 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.

OCR levels were measured using a Cell Mito Stress Test Kit on a Seahorse XF96 analyzer (Agilent) according to manufacturer’s instructions. Briefly, cells were irradiated and plated at 20,000 cells/well in XF96-well plates. The next day, cells were washed and equilibrated with buffer-free medium (DMEM + Glutamax with 10 mM glucose and 2 mM L-glutamine) at 37°C in a CO₂-free incubator for 1 h. Initial measurements of ECAR and OCR were collected. Cells were then consecutively treated with 1 μM oligomycin, 1 μM FCCP and 0.5 μM rotenone/antimycin A, during which OCR values were assessed. Data were normalized to cell numbers obtained by counting cell nuclei after paraformaldehyde fixation and DAPI staining.

To further evaluate the metabolism levels of GC cells, ECAR and OCR were determined with a Glycolysis Stress Test Kit and Cell Mito Stress Test Kit (Agilent, USA). Briefly, cells were seeded in Seahorse XF96 plates (Agilent). After 10 h of incubation, the ECAR and OCR were detected according to the manufacture’s protocols. In the detection of ECAR, glucose (10 mM), oligomycin (1 μM) and 2-deoxy-d-glucose (100 mM) were injected into medium of cells sequentially. For the measurement of OCR, cells were treated with oligomycin (1 μM), protonophore trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, 0.5 μM) and antimycin A (0.5 μM). The signaling data were read and recorded by a Seahorse XFe96 Analyzer (Agilent).

Extracellular acidification rates (ECAR) and oxygen consumption rate (OCR) were measured using a Seahorse XFe96 (Agilent Technologies, Santa Clara, CA, USA) with the Seahorse XF Glycolysis Stress Test Kit (cat no. 103020‐100, Agilent Technologies) and Cell Mito Stress Test Kit (cat no. 103015‐100, Agilent Technologies), according to the manufacturer's instructions. In brief, 5 × 10⁴ cells were seeded into XF96 culture plates in quintuplicate 1 day before analysis. ECAR was measured under basal conditions and after sequential treatment of the PAAD cells with serial injections of glucose (10 mmol/L), oligomycin (1 μmol/L) and 2‐deoxy‐D‐glucose (2‐DG) (100 mmol/L). For OCR measurement, 1 μmol/L oligomycin, 1 μmol/L carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone and 1 μmol/L rotenone were automatically injected into XF96 Cell Culture Microplates. ECAR and OCR were normalized to the cell number as determined by CellTiter‐Glo analysis at the end of the experiments.

For the assessment of mitochondrial function, a Cell Mito Stress Test Kit (Agilent) was used to assay the oxygen consumption rate (OCR) on a Seahorse XFp Extracellular Flux Analyzer (Agilent). S + O + Z-transduced ADSCs and neural stem cell (NSC)-OPCs were seeded at 20,000 cells/well in poly-L-ornithine + laminin-coated Seahorse XFp Cell Culture Miniplates (Agilent) and maintained for 24 h in proliferative growth medium (NBB27 medium supplemented with EGF + bFGF + PDGF-AA; for converted cells, doxycycline was also added).
On the day of the experiment, the medium was changed to Seahorse XF DMEM, pH 7.4 supplemented with 1 mM pyruvate, 25 mM glucose, and 4 mM L-glutamine, and the plates were incubated for 1 h at 37°C without CO₂. The assay is based on optical sensors that measure the oxygen concentration after the sequential addition of drugs that modulate cellular respiration (1 µM oligomycin, which blocks ATP synthase; 2 µM FCCP, a protonophore that enables maximum electron flux through the electron transport chain; and rotenone and antimycin A [RAA], which inhibit complexes I and III, respectively, thus shutting down mitochondrial activity), allowing to estimate the basal, ATP-linked, or maximal capacity of mitochondrial respiration.

A Seahorse XF96 analyzer (Seahorse Biosciences, Billerica, Massachusetts, USA) was used to assess the oxygen consumption rate (OCR), which reflects the rate of OXPHOS. A total of 15 000 cells were seeded into a 96‐well XF96 plate and cultured overnight. The Cell Mito Stress Test Kit (Agilent Technologies Inc., Santa Clara, California, USA) was used to measure cellular mitochondrial flux. Several inhibitors, oligomycin (1.25 μm, mitochondrial ATP synthase inhibitor), carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP) (2.5 μm, protonophore and uncoupler of ATP synthesis from mitochondrial respiration), and rotenone (0.75 μm, electron transport inhibitor) were added according to the manufacturer's instructions. ATP production and spare respiratory capacity were two important indicators to reflect the ability of OXPHOS. At basal respiration, OCR decreased significantly after oligomycin was added to inhibit ATPase; reducing OCR was represented to the ability of ATP production. After adding the FCCP uncoupling agent, electron transport would lose the constraint of proton gradient and move at the maximum speed, enabling OCR to increase sharply to the maximal oxygen consumption. At this time, the difference between the maximal oxygen consumption and basic respiration is called spare respiratory capacity.