SVFs from inguinal fat pads were isolated as described above and were seeded in an XF24 V28 microplate (Seahorse Bioscience) coated with poly-l -lysine (Sigma), followed by differentiation into beige adipocytes in the cocktail as described above with treatment of rat PTH1–84 or Vehicle for five days. Subsequently, the OCR was measured as previously described [18 (link)] using an XF24 analyzer (Seahorse Bioscience) according to manufacturer's instructions. Briefly, the culture medium of the cells was changed to Seahorse XF base medium with 25 mM d -glucose, 2 mM sodium pyruvate, and 2 mM LG and incubated at 37 °C in a non-CO2 incubator (Seahorse Bioscience) for one hour. Respiratory inhibitors (Seahorse Bioscience, 103,015–100) including 1 mg/ml oligomycin, 1.5 mM FCCP, 0.5 mM antimycin A, and 0.5 mg/ml rotenone were loaded into the injection port in sequence and to detect the base respiration, uncoupled respiration, maximal respiration, and non-mitochondrial respiration, respectively. The final OCR results were standardized to the protein concentration.
Xf24 v28 microplate
Manufactured by Agilent Technologies
Sourced in United States
The XF24 V28 microplate is a laboratory equipment product designed for cellular analysis. It provides a platform for measuring key parameters of cellular metabolism, including oxygen consumption rate and extracellular acidification rate. The microplate is compatible with Agilent's XF Analyzer instruments and is optimized for high-throughput analysis of cellular samples.
Automatically generated - may contain errors
Lab products found in correlation
Xf24 analyzer, by Agilent Technologies (4 mentions)
Non co2 incubator, by Agilent Technologies (3 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Seahorse xf base medium, by Agilent Technologies (1 mentions)
Mitostress kit, by Agilent Technologies (1 mentions)
Seahorse xf24, by Agilent Technologies (1 mentions)
5 protocols using xf24 v28 microplate
1
Beige Adipocyte Oxygen Consumption Assay
2
Beige Adipocyte Oxygen Consumption Assay
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SVFs were seeded in an XF24 V28 microplate (Seahorse Bioscience) coated with poly-L-lysine. After 24 h, the cells were infected with lentivirus and subsequently differentiated into beige adipocytes for five days. Oxygen Consumption Rate (OCR) was measured as previously described (Wang et al., 2013 (link)) using an XF24 analyzer (Seahorse Bioscience) in accordance with the manufacturer's instructions. In brief, the cells were washed with Seahorse XF base medium with 25 mM D-glucose, 2 mM sodium pyruvate, and 2 mM LG, followed by incubation with 525 μl assay medium with or without 2% fatty acid-free BSA at 37 °C in a non-CO2 incubator (Seahorse Bioscience) for 1 h. 75 μl respiratory inhibitors (Seahorse Bioscience, 103015-100) was loaded into the injection port to reach the final concentration of 1 mg/ml oligomycin, 1.5 mM FCCP, 0.5 mM antimycin A and 0.5 mg/ml rotenone to detect the uncoupled respiration, maximal respiration, and non-mitochondrial respiration, respectively. The final OCR results were standardized to the protein concentration. The results are representative of at least three independent experiments.
3
Measurement of Adipocyte Respiration
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SVFs were seeded in an XF24 V28 microplate (Seahorse Bioscience) precoated with poly-l -lysine and subsequently induced to differentiate into brown or beige adipocytes. At day 4 of differentiation, the oxygen consumption rate (OCR) was measured as previously described using an XF24 analyzer with the Mito stress kit (Agilent, 103,015) in accordance with the manufacturer's instructions [25 (link)]. Briefly, the cells were washed and incubated with prewarmed Seahorse XF base medium with 25 mM glucose, 2 mM sodium pyruvate, and 2 mM glutamine (pH 7.4) in a non-CO2 incubator (Seahorse Bioscience) at 37 °C for 1 h. The drug injection ports of the sensor cartridge were loaded with 75 μl respiratory inhibitors, and all following measurements were conducted for three cycles. After measuring basal OCR, 2 μM oligomycin, 1 μM FCCP, or 1 μM rotenone/1 μM antimycin was added to measure uncoupled respiration, maximal respiration, and non-mitochondrial respiration, respectively. The initial OCR values were automatically calculated by Seahorse XF24 software (Wave, Seahorse Bioscience), and the final OCR results were standardized to total protein content in each well.
4
Measuring Oxygen Consumption in Induced Brown/Beige Adipocytes
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SVFs were seeded in an XF24 V28 microplate (Agilent Technologies, USA) coated with poly-L-lysine. The induction protocol was as described in section 2.5. The oxygen consumption rate (OCR) was measured at induction day 4 using an XF24 analyzer (Agilent Technologies) following the manufacturer’s instructions. Briefly, the induced brown/beige adipocytes were washed with Seahorse assay medium, consisting of XF DMEM supplemented with 10 mM XF glucose, 1 mM XF pyruvate, and 2 mM XF L-glutamine, followed by incubation with 525 μl of assay medium at 37°C in an incubator without CO2 (Agilent Technologies) for 1 h. Respiratory inhibitors (75 μl) were loaded into the injection port to final concentrations of 1 mg/ml oligomycin, 2 mM FCCP, 0.5 mM antimycin A, and 0.5 mg/ml rotenone to detect uncoupled respiration, maximal respiration, and nonmitochondrial respiration, respectively. The final OCR results were standardized to the total protein content. The results are representative of at least three independent experiments.
5
Measuring Brown Adipocyte Respiration
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SVFs were seeded in an XF24 V28 microplate (Agilent Technologies, USA) coated with poly-L-lysine, and induced to mature brown adipocytes as previously mentioned.
The oxygen consumption rate (OCR) was measured on the fourth day of induction using an XF24 analyzer (Agilent Technologies) following the manufacturer's instructions. Briefly, the induced brown adipocytes were washed with Seahorse assay medium, consisting of XF DMEM supplemented with 10 mM XF glucose, 1 mM XF pyruvate, and 2 mM XF L-glutamine, followed by incubation with 525 ml of assay medium at 37°C in an incubator without CO 2 (Agilent Technologies) for 1 h.
Respiratory inhibitors (75 ml) were loaded into the injection port with the final concentrations of 1 mg/ml oligomycin, 2 mM FCCP, 0.5 mM antimycin A and 0.5 mg/ml rotenone to detect uncoupled respiration, maximal respiration, and nonmitochondrial respiration, respectively. The final OCR results were standardized to the total protein content per well.
The oxygen consumption rate (OCR) was measured on the fourth day of induction using an XF24 analyzer (Agilent Technologies) following the manufacturer's instructions. Briefly, the induced brown adipocytes were washed with Seahorse assay medium, consisting of XF DMEM supplemented with 10 mM XF glucose, 1 mM XF pyruvate, and 2 mM XF L-glutamine, followed by incubation with 525 ml of assay medium at 37°C in an incubator without CO 2 (Agilent Technologies) for 1 h.
Respiratory inhibitors (75 ml) were loaded into the injection port with the final concentrations of 1 mg/ml oligomycin, 2 mM FCCP, 0.5 mM antimycin A and 0.5 mg/ml rotenone to detect uncoupled respiration, maximal respiration, and nonmitochondrial respiration, respectively. The final OCR results were standardized to the total protein content per well.
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