Xf24 analyser

An XF24 Analyser (Agilent Technologies, Santa Clara, CA, USA) was used to measure the respiratory function of primary osteoblasts. Osteoblasts were plated at a density of 50,000 cells per well and transferred to a 37 °C CO₂ incubator until and calvarial osteoblasts were differentiated using osteogenic differentiation media containing 8 mM β-glycerophosphate and 50 μg/ml ascorbic acid for 3 days. On the day of the assay, cells were washed in XF Assay Media supplemented with 25 mM glucose and 10 mM pyruvate and placed in a non-CO2 incubator at 37 °C for 1 h prior to start of assay. Reagents were prepared for the assay (injection volume of 75 μL for each reagent per well) from 2.5 mM Seahorse stock solutions, Oligomycin (1.2 μM). Following equilibration, the Seahorse plate was placed in the Seahorse XF24 Analyser for sample analysis. The raw data was normalised to protein content in each well at the end of the assay [71 (link)].

The XF Mitostress assay (Agilent Technologies, UK) was performed as previously described [84 (link)]. Briefly, hepatic mitochondria were isolated and added to an XF culture plate at a concentration of 10 μg per well. A pre-soaked eXF cartridge was prepared [89 (link)], the culture plate loaded into an XF24 Analyser (Agilent Technologies, UK) and the assay initiated. Oxygen consumption rate (OCR) was measured in substrate alone (10 mM pyruvate, 2 mM malate), during state 3 (ADP [4 mM]), state 4 (oligomycin [2.5 μg/ml]), state 3u (FCCP (carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone) [4 μM]), and lastly antimycin A with rotenone [both 4 μM]. All reagents were sourced from Sigma, UK. Superoxide dismutase (SOD) and protein carbonyl activities were quantified in liver tissue homogenates using Total SOD activity assay and Carbonyl assay kits, respectively in line with the manufacturer’s guidelines (Cayman Chemical Company, Estonia).

A Seahorse Bioscience XF24 analyser (Agilent Technologies, Santa Clara, CA, USA) was used to measure the mitochondrial O₂ consumption rate (OCR) [37 (link),38 (link)]. Cells were seeded in the specific 24-well plates (20,000 per well). Before each measurement, the cells were washed with PBS, and 590 µL of Agilent Seahorse XF Base Medium (without phenol red and bicarbonate) supplemented with 1 mM pyruvate and 25 mM glucose was added to each well. Measurements were normalised according to protein concentration (Bradford, England). For OCR experiments, we used the commercial “Seahorse XF cell Mito Stress test kit” (Agilent Technologies, USA) following the manufacturer’s instructions; this kit uses oligomycin, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and rotenone/antimycin as mitochondrial toxins. The sequence of mitochondrial toxins added was oligomycin, 1 µM; FCCP, 0.5 µM; rotenone/antimycin A, 0.5/0.5 µM. The inclusion of rotenone/antimycin A served to measure respiration by non-mitochondrial processes, as these compounds eliminate mitochondrial respiration, which was then subtracted from all OCR values obtained.

Mitochondrial bioenergetics were measured in L6 myoblasts using a Seahorse XF24 analyser. WT L6 muscle cells or those that had been genetically manipulated as specified in the figure legends were seeded and cultured in 24‐well Seahorse culture plates and switched to phenol‐ and serum‐free Dulbecco's modified Eagle's medium (DMEM) (standard AA formulation [#D5030‐10L]) containing 5 mM of glucose, 2 mM of glutamine and 10 mM of HEPES prior to analysis of basal respiration, protein synthesis‐dependent respiration, ATP‐linked respiration and non‐mitochondrial respiration (using cycloheximide, oligomycin, rotenone and antimycin). Live cell respiration parameters were normalized to cell protein.

An XF24 Analyser (Seahorse Biosciences, Billerica, MA) was used to measure metabolic changes in b.End3 cells [10] (link), [41] (link), [42] (link). The XF24 creates a transient 7 μl chamber in specialised microplates that allows real-time measurement of oxygen and proton concentration changes via specific fluorescent dyes and calculates OCR (oxygen consumption rate) and PPR (proton production rate), measures of mitochondrial respiration and glycolytic activity. The proton production rate is expressed in pMol/min, while ECAR is in pH/min. The OCR and PPR values represent the metabolism of cells, but may also reflect the number of viable cells.
b.End3 cells were exposed to hyperglycemia for 7 days and treated with H₂S donors for 3 days as described above. The culture medium was changed to unbuffered DMEM (pH 7.4) containing 5 mM glucose, 2 mM L-glutamine and 1 mM sodium pyruvate to allow measurement of the proton production. After determining the basal OCR and PPR values, oligomycin, FCCP and antimycin A were injected through the ports of the Seahorse Flux Pak cartridge to reach final concentrations of 1 μg/ml, 0.3 μM and 2 μg/ml, respectively, to determine the amount of oxygen consumption linked to ATP production, the level of non-ATP-linked oxygen consumption (proton leak) as well as the maximal respiration capacity and the non-mitochondrial oxygen consumption.

Oxygen consumption was measured on a Seahorse XF24 analyser at 37°C. Seahorse XFe24 FluxPak mini (102342-100) and Seahorse XF base medium (102353-100) was purchased from Agilent (California). Oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and Rotenone/Antimycin-A were purchased from Sigma. Forty-eight hours before the assay, 40,000 siSRSF3 or siNeg HepG2 cells were seeded in a Seahorse XF24 analyser plate. After inducing enough lipid droplets by pretreating HepG2 cells with 0.2 mM PA for 24 h, we replaced the medium with Seahorse XF base medium and measured the mitochondrial respiration in the basal state and after 1 μM Oligomycin, 2 μM FCCP, and 0.5 μM Rotenone/Antimycin-A treatment. Further, in another assay, instead of pretreating cells with PA to produce lipid droplets, we added excessive exogenous PA and then measured oxygen consumption. Adenosine triphosphate (ATP) production, basal respiration, maximal respiration, and spare respiratory capacity were calculated, respectively. Cells were lysed and protein concentration was taken for normalization.

The effects of DM-AKG on mitochondrial oxidative phosphorylation in RANKL-stimulated RAW264.7 cells were assessed by the XF24 Analyser and XF Cell Mito Stress Test Kit (Seahorse Biosciences, North Billerica, MA, USA), as described previously [27 (link)]. Briefly, following the treatment, the culture medium was substituted with the designated test medium. During the test, the oligomycin (0.5 μM), carbonyl cyanide 4-(trifluoromethoxy), phenylhydrazone (FCCP, 1.0 μM), and rotenone/antimycin A (1.0 μM) were sequentially injected into the test medium. Measurement of basic respiration, maximal respiration capacity, ATP production, and proton leak were obtained through the probes. The total protein content of the cells was measured in order to normalize the final data. Seahorse XF Report Generator software (Wave, Agilent) was utilized to analyze the measurement results.