The amounts of glycerol and extracellular lactate released to the culture medium were determined using lipolysis and lactate colorimetric assay kits (BioVision), respectively. To measure OCR, we used the oxygen consumption rate assay kit (Abcam). In these assays, we plated 15,000 cells per well in 96-well plates, proceeded with the adipogenic differentiation protocol, and, finally, performed the assays following the manufacturers’ recommendations. Colorimetric determinations to quantify extracellular lactate in the culture medium and the release of glycerol to the medium were performed with a Plate Reader Axis UVM340 (Biochrom). To measure the OCRs of the different cultured adipocytes, we used an F-7000 fluorescence spectrophotometer (Hitachi). Data were normalized to the amount of total protein in each well.
Oxygen consumption rate assay kit
Manufactured by Abcam
Sourced in United States
The Oxygen Consumption Rate Assay Kit is a tool used to measure the rate of oxygen consumption in cells or tissue samples. It provides a quantitative assessment of cellular respiration and mitochondrial function.
Automatically generated - may contain errors
Lab products found in correlation
Lactate colorimetric assay kit, by Abcam (1 mentions)
F 7000 fluorescence spectrophotometer, by Hitachi (1 mentions)
Plate reader axis uvm340, by Harvard Bioscience (1 mentions)
Seahorse xfe96 analyzer, by Agilent Technologies (1 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
Lactate fluorometric assay kit, by Abcam (1 mentions)
Cell mitochondria isolation kit, by Beyotime (1 mentions)
Z yvad fmk, by Merck Group (1 mentions)
Cell counting kit 8 (cck8), by Beyotime (1 mentions)
Mcc950 sc 505904, by Santa Cruz Biotechnology (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Mitochondria storage buffer, by Beyotime (1 mentions)
4 protocols using oxygen consumption rate assay kit
1
Quantification of Adipocyte Metabolism
2
Extracellular Oxygen Consumption Assay
3
Metabolic Profiling of 12Z Cells under PV Treatment
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The oxygen consumption rate and lactate production in 12Z cells were measured after 4 h of PV treatment to clarify the influence of PV on metabolic activity. First, the oxygen consumption rate was analyzed using an oxygen consumption rate assay kit (Abcam, Cambridge, MA, United States). 12Z cells were seeded in a black 96-well microplate at a density of 3 × 104 cells. The next day, various concentrations of PV (0, 62.5, 125, and 250 μg/ml) were added, and the experiment was conducted according to the manufacturer’s instructions. Second, lactate production was measured using a lactate fluorometric assay kit (Biovision, CA, United States). After 4 h of PV treatment, the media was replaced with phenol red- and serum-free DMEM and incubated for 1 h at 37°C. Then, 1 μl of the medium from each well was assessed by fluorescence at Em 535/Ex 590 nm using the Spectramax M2 spectrofluorometer. Dichloroacetate (DCA) was used as a positive control for lactate production.
4
Mitochondrial Dysfunction and Inflammasome Regulation
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Neu-p11 (Piromelatine) was purchased from Neurim Pharmaceuticals Ltd. (Tel-Aviv, Israel); MCC950 (sc-505904) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Z-YVAD-FMK (235429) was purchased from Sigma-Aldrich (Sigma, St Louis, MO, USA). GKT137831 (B4763) was purchased from ApexBio Technology (ApexBio, Houston, Texas, USA). Oxygen Consumption Rate Assay Kit (ab197243) was purchased from Abcam (Cambridge, UK). Mito-SOX Red Mitochondrial Superoxide Indicator) (M36008) was purchased from Invitrogen (Carlsbad, CA, USA). Cell Counting Kit-8 (C0037), LDH Cytotoxicity Assay Kit (C0016), Mitochondrial membrane potential assay kit with JC-1 (C2006), Caspase 1 activity assay kit (C1101), Cell Mitochondria Isolation Kit (C3601) and Mitochondria Storage Buffer (C3609) were purchased from Beyotime (Nanjing, PRC). The antibodies used in this study are: β-actin (ab179467), NOX4 (ab133303), NLRP3 (ab232401), ASC (ab155970), pro-Caspase-1 (ab179515), PARP (ab74290), MnSOD (ab13533), Sirt3 (ab223531), Tfam (ab131607), COX IV (ab33985), Donkey Anti-Goat IgG H&L(Cy5) (ab6566), Donkey Anti-Rabbit IgG H&L(HRP) (ab6802) were purchased from Abcam (Cambridge, UK). Cleaved-Caspase-1(D57A2) was purchased from Cell Signaling Technology (Beverly, MA, USA); pro-IL-1β (AF-401) was purchased from R&D Systems (Minneapolis, USA).
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