Oxygen consumption rate (OCR) was measured in NRCMs transfected with control siRNA, GCN5L1 siRNA, control adenovirus, or GCN5L1 adenovirus using the Seahorse XF system (Seahorse Bioscience) as described previously.20 (link) Basal OCR in each well was measured, followed by serial treatment with FCCP (7.5 μmol/L) and rotenone (2 μmol/L). Each experiment was repeated to ensure reproducibility, and the data presented are technical replicates (N = 8-11) of a single representative study. NRCMs were transfected with GCN5L1 siRNA (Origene # SR15522) and control siRNA using Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific #13778150) according to the manufacturer’s protocol. The adenovirus overexpressing GCN5L1 was previously reported.29 (link) Briefly, adenoviruses were produced using the Adeasy Adenoviral system (Agilent), and a multiplicity of infection (MOI) of 10 was used for all the experiments. NRCMs were treated with phenylephrine (20 μM; Sigma #P6126), 24 hours after siRNA transfection, for 48 hours before use or harvest. The human TFAM WT, 4KQ (K62Q/K76Q/K111Q/K118Q), and 4KR (K62R/K76R/K111R/K118R) plasmids were custom synthesized by Genscript, and transiently transfected for 48 hours into AC16 cells using Lipofectamine as above.
Seahorse xf system
Manufactured by Agilent Technologies
The Seahorse XF system is a laboratory instrument designed to measure the metabolic activity of live cells in real-time. It provides a comprehensive analysis of cellular respiration and glycolysis, allowing researchers to gain insights into cellular bioenergetics and metabolism.
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2 protocols using seahorse xf system
1
Mitochondrial Bioenergetics in Cardiomyocytes
2
Oxygen Consumption Assay in H9c2 Cells
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Oxygen consumption rate (OCR) was measured in GCN5L1 control and knockdown stable H9c2 cell lines using the Seahorse XF system (Seahorse Bioscience). Cells (5 × 103 cells/well) in each condition were plated and allowed to attach overnight in DMEM. On the assay day, media was changed to unbuffered DMEM media supplemented with either 5 mmol/L glucose (low glucose) or 25 mmol/L glucose (high glucose), 2 mmol/L glutamine and 0.5 mmol/L carnitine, and the plate incubated in a non‐CO2 incubator (37°C; 15 min) before running. Basal OCR in each well was measured, followed by serial treatment with FCCP (7.5 μmol/L) and rotenone (2 μmol/L). After completion, viability was assessed by crystal violet (CV) staining, and OCR normalized to cell number. Each experiment was repeated to ensure reproducibility, and the data presented are technical replicates (N = 8) of a single representative study.
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