ECAR and oxygen consumption rate (OCR) values were measured by the Agilent Seahorse XFe96 instrument at the University of Pennsylvania Diabetes Research Center, Islet Cell Biology Core. Base XF media, extracellular flux assay plates, cell culture plates, and mitochondrial stress test kits were purchased from Agilent. All procedures were performed according to the manufacturer’s instructions and as previously described (Traba et al., 2016 (link)). cell culture plates were coated overnight with CellTak (Corning) and allowed to air-dry. FO B cells were isolated from WT and Itch KO mice and stimulated in vitro overnight, and then 250,000 cells per well were plated onto CellTak-coated plates in mitochondrial stress test media supplemented with stimulation reagents (e.g., anti-IgM and CpG). ECAR and OCR were measured before and after treatment with oligomycin, and data were analyzed by Wave software (Agilent). Basal glycolysis was ECAR before adding oligomycin, and max glycolysis was ECAR after treatment with oligomycin. Non–electron transport chain oxygen consumption was OCR after addition of oligomycin. Basal oxidative respiration was OCR before adding oligomycin.
Cell culture plates
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Cell culture plates are a fundamental piece of laboratory equipment used for in vitro cell culture experiments. They provide a sterile, flat surface with multiple wells to cultivate cells in a controlled environment.
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Lab products found in correlation
2 protocols using cell culture plates
1
Mitochondrial Respiration of B Cells
2
Podocyte Metabolic Function Analysis
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The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of podocytes were assessed by an Agilent Seahorse XF extracellular flux analyzer (Agilent Technologies) according to our previous description (16) . Briefly, podocytes were seeded into cell culture plates (Agilent Technologies). Data were calculated from three independent measurements obtained prior to or after compound injection.
MMP and NAD + Content Measurements MMP was measured using the JC-1 mitochondrial potential sensor (Invitrogen) according to the manufacturer's protocols. Podocytes were harvested and incubated with JC-1 solution, and the fluorescence of JC-1 monomers and J-aggregates was analyzed by flow cytometry. NAD 1 content was measured using an NAD 1 Assay Kit (Beyotime Biotechnology) according to the manufacturer's recommendation.
MMP and NAD + Content Measurements MMP was measured using the JC-1 mitochondrial potential sensor (Invitrogen) according to the manufacturer's protocols. Podocytes were harvested and incubated with JC-1 solution, and the fluorescence of JC-1 monomers and J-aggregates was analyzed by flow cytometry. NAD 1 content was measured using an NAD 1 Assay Kit (Beyotime Biotechnology) according to the manufacturer's recommendation.
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