Mitochondrial function was determined in intact cells by real-time respirometry (Seahorse XFe24; Agilent). MDA-MB-231 and EO771 cells were seeded at 1.0 × 104 cells/well in an XFe 24-well plate (Agilent). Once cells reached 80% confluency, they were treated with a vehicle (0.01% DMSO) or varying concentrations of BAM15 for 16 h. Following treatment, media was removed, and cells were incubated at 37°C for 1 h in XF DMEM medium (pH 7.4) supplemented with 1 mM pyruvate and 2 mM glutamine without CO2. Cells were then serially injected with 10 mM glucose, 1 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone and antimycin A. Components of mitochondrial function were calculated as described previously [67 (link)]. Data were normalized to nuclear content by staining live cells after assay with 20 μM Hoechst 33342 (ThermoFisher Scientific) and reading fluorescence on an automated microplate reader (Biotek) at excitation/emission 350/461 nm. Mitochondrial respiration was quantified as described previously [18 (link)].
Xfe 24 well plate
Manufactured by Agilent Technologies
The XFe 24-well plate is a laboratory equipment product designed for use in Agilent's Seahorse XFe Analyzer. The plate features 24 individual wells, providing a platform for measuring cellular metabolic activity and function.
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4 protocols using xfe 24 well plate
1
Mitochondrial Respiration Profiling in Cells
2
Mitochondrial Function Evaluation in C2C12 Myotubes
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Intact cell mitochondrial function was assessed by real‐time respirometry (Seahorse XFe24; Agilent). C2C12 myoblasts were seeded at 10,000 cells/well in an XFe 24‐well plate (Agilent). After 4 days of differentiation, myotubes were treated with varying concentrations of BAM15 for 16 h. Following treatment, media were removed and cells were incubated for 1 h in XF DMEM (pH 7.4) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose at 37°C, without CO2. Cells were then serially injected with 1 μM oligomycin, 1 μM FCCP, and 0.5 μM of rotenone and antimycin A. Components of mitochondrial function were calculated as described previously (Brand & Nicholls, 2011 ). Data were normalized to protein content by BCA assay (Thermo Scientific).
3
Quantifying Cellular Respiration Dynamics
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Oxygen consumption dynamics were assessed by real‐time respirometry (Seahorse XFe24; Agilent). C2C12, AML12, and 3T3‐L1 cells were seeded at 10,000 cells/well in an XFe 24‐well plate (Agilent). Following expansion and differentiation, media were removed and cells incubated for 1 h in XF DMEM (pH 7.4) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose at 37°C, without CO2. Cells were then injected with 1 μM of BAM15, DNP, or FCCP, and the rates of oxygen consumption (OCR) and extracellular acidification (ECAR) were measured over 12 h. Data were normalized to protein content by BCA assay (Thermo Scientific). Maximal OCR was defined as the greatest rate achieved over the 12‐h period. Time to maximal OCR was determined by subtracting the time of maximal OCR from baseline injection. The respiratory half‐life (t1/2) was defined as the time by which the OCR had decayed to 50% of the maximal observed respiration.
4
Measuring Cellular Oxygen Dynamics
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Oxygen consumption dynamics were assessed by real-time, intact cell respirometry (Seahorse XFe24; Agilent). MDA-MB-231 and EO771 cells were seeded at 1.0 × 104 cells/well in an XFe 24-well plate (Agilent). Following expansion, media was removed, and cells were incubated at 37°C for 1 h in XF DMEM medium (pH 7.4) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose without CO2. Cells were then injected with 1, 10, or 20 μM of BAM15 or vehicle and the rates of oxygen consumption (OCR) and extracellular acidification (ECAR) were measured over 12 h. Data were normalized to nuclear double-stranded DNA content by staining live cells after assay with 20 μM Hoechst 33342 (ThermoFisher Scientific, Waltham, MA, USA) and reading fluorescence on an automated microplate reader (Biotek) at excitation/emission 350/461 nm. Maximal OCR was defined as the greatest rate achieved over the 12-h period. Time to maximal OCR was determined by subtracting the time of maximal OCR from baseline injection. The respiratory half-life (t1/2) was defined as the time by which the OCR had decayed to 50% of the maximal observed respiration as described previously [18 (link)].
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