Cell mito stress kit

Manufactured by Agilent Technologies

Sourced in United States

The Cell Mito Stress Kit is a laboratory equipment product designed to measure mitochondrial function in cells. It provides tools to assess key parameters of cellular bioenergetics, including oxygen consumption rate, extracellular acidification rate, and mitochondrial respiration.

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25 protocols using cell mito stress kit

Evaluating Nebivolol's OXPHOS Inhibition

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To confirm the OXPHOS inhibitory function of Nebivolol, SUM159 TNBC cells (15,000 cells/well) were seeded onto XFp Seahorse cell plate and treated with 10 μM nebivolol. The oxygen conception rate (OCR) was measured using Cell Mito Stress kit (Cat. 103010-100, Agilent Technologies) in a Seahorse XFp Extracellular Flux Analyzers (Agilent Technologies) according to manufacturer’s instructions and as described before (Park et al., 2016 (link)).

Abdulkareem N.M., Bhat R., Powell R.T., Chikermane S., Yande S., Trinh L., Abdelnasser H.Y., Tabassum M., Ruiz A., Sobieski M., Nguyen N.D., Park J.H., Johnson C.A., Kaipparettu B.A., Bond R.A., Johnson M., Stephan C, & Trivedi M.V. (2022). Screening of GPCR drugs for repurposing in breast cancer. Frontiers in Pharmacology, 13, 1049640.

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Mitochondrial Function and Glycolysis

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Mitochondrial function (oxygen consumption rate) and glycolysis (acidification rate) of BMDMs were measured with a Seahorse XF24 extracellular flux analyzer using the Cell Mito Stress Kit (Agilent Technologies) according to the manufacturer’s instructions.

Pfefferlé M., Ingoglia G., Schaer C.A., Hansen K., Schulthess N., Humar R., Schaer D.J, & Vallelian F. (2021). Acute Hemolysis and Heme Suppress Anti-CD40 Antibody-Induced Necro-Inflammatory Liver Disease. Frontiers in Immunology, 12, 680855.

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Bioenergetic Profile of Intact Neurons

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The Seahorse XF24 Extracellular Flux Analyzer (Agilent Technologies) was used to determine the bioenergetic profile of intact neurons (Lomeli et al., 2017 ). Oxygen consumption rates (OCR) were measured in adherent hippocampal neurons using the Cell Mito Stress Kit (Agilent Technologies). Dissociated hippocampal neurons were plated at a density of 5 × 10⁴ cells/well in 0.1 mg/mL poly-D-lysine coated XF24 cell culture microplates. Baseline rates were measured at 37 °C three times before the sequential injection of oligomycin (2 μM), FCCP (1 μM), and rotenone (1 μM) plus antimycin A (1 μM). Basal OCR levels were determined by subtracting the non-mitochondrial respiration rate from the last rate measurement before the oligomycin injection. Maximum respiration was calculated by subtracting the non-mitochondrial respiration rate from the maximum rate measurement after FCCP injection. Three OCR measurements were taken after the addition of each inhibitor. All measurements were normalized to protein content per well using the DC protein assay (Bio-Rad). OCR data was collected using the Wave software (Agilent Technologies) and analyzed using the XF Cell Mito Stress Kit Report generator.

Lomeli N., Di K., Pearre D.C., Chung T.F, & Bota D.A. (2020). Mitochondrial-associated impairments of temozolomide on neural stem/progenitor cells and hippocampal neurons. Mitochondrion, 52, 56-66.

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Mitochondrial Respiration Assay

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Oxygen consumption rate was assayed using Cell Mito Stress kit (Agilent) and analyzed on Seahorse XF HS mini analyzer.

Caplan M., Wittorf K.J., Weber K.K., Swenson S.A., Gilbreath T.J., Willow Hynes-Smith R., Amador C., Hyde R.K, & Buckley S.M. (2022). Multi-omics reveals mitochondrial metabolism proteins susceptible for drug discovery in AML. Leukemia, 36(5), 1296-1305.

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Mitochondrial Respiration Assay

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Oxygen consumption rate was assayed using Cell Mito Stress kit (Agilent) and analyzed on Seahorse XF HS mini analyzer.

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Mitochondrial Respiration in NSC34 Cells

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XF DMEM medium, XF24 culture plates, XF24 FluxPak, and a Cell Mito Stress kit were purchased from Agilent (Santa Clara, CA, USA). NSC34 cells were seeded in the XF24 plates at 5~10 × 10³ cells per well, transfected with plasmid pcDNA-mt-SOD1^G93A-GFP, and then treated with 1 mM butyrate, as described in the text. The oxygen consumption rate (OCR) of cultured cells was determined with an Agilent Seahorse XFe24 Analyzer, following the manufacturer’s instructions. After the assay, the cells were immediately stained with Hoechst33324 (ENZO, New York, NY, USA), staining the nucleus). The whole wells of stained cells were imaged through the tile scan method under a Leica DMi8 microscope. The stitched images were imported into ImageJ for background subtraction, thresholding, binarization, and cell counting.

Li X., Dong L., Li A., Yi J., Brotto M, & Zhou J. (2022). Butyrate Ameliorates Mitochondrial Respiratory Capacity of The Motor-Neuron-like Cell Line NSC34-G93A, a Cellular Model for ALS. Biomolecules, 12(2), 333.

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Extracellular Flux Analysis of Cellular Respiration

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Cells were cultured in RPMI-1640 medium without phenol red for three days, followed by harvesting, resuspension in the assay medium (Agilent, 103576-100) and plating in the XF24 assay plate (Agilent, 100777-004) at a density of 0.1 million cells per well. The cells were then kept at 37°C without CO₂ for 1 h. Oxygen consumption rate (OCR) was measured using Cell Mito Stress Kit (Agilent, 103015-100) with the following sequential injection of mitochondria inhibitors: 1.5 μM oligomycin, 1 μM Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and then 0.5 uM of Antimycin A and Rotenone. Extracellular acidification rate (ECAR) was measured using Glycolysis Stress Kit (Agilent, 103020-100), with cells metabolically perturbed by sequential injections of 10mM glucose, 1 μM oligomycin and 50 mM 2-deoxyglucose. OCR and ECAR levels were recorded using a Seahorse XF-24 extracellular flux analyzer following manufacturer's instructions (Seahorse Biosciences).

Xu C., Tsai Y.H., Galbo PM J.r., Gong W., Storey A.J., Xu Y., Byrum S.D., Xu L., Whang Y.E., Parker J.S., Mackintosh S.G., Edmondson R.D., Tackett A.J., Huang J., Zheng D., Earp H.S., Wang G.G, & Cai L. (2021). Cistrome analysis of YY1 uncovers a regulatory axis of YY1:BRD2/4-PFKP during tumorigenesis of advanced prostate cancer. Nucleic Acids Research, 49(9), 4971-4988.

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Mitochondrial Function Assessment of hPSC-CMs

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hPSC-CMs were seeded overnight onto Geltrex (Thermo Fisher Scientific) coated microplates (part of the Seahorse XF flux pack) at a density of 30,000 per well of a 96 well plate. One hour prior to measurement, medium was replaced with Seahorse XF DMEM medium supplemented with Seahorse XF Glucose (10 mM), Pyruvate (1 mM) and L-Glutamine (2 mM) (Agilent) and cells were incubated at 37°C in an incubator without CO₂ for 1 h. The Cell Mito Stress kit (Agilent) was used to assess mitochondrial function. In brief, oxygen consumption rate was measured at baseline, upon oligomycin (2.5 μM), FCCP (2. μM) and rotenone/antimycin (1 μM) injections (Agilent, 103015-100) using the Seahorse XFe96 Analyzer. Data were normalized to the number of cells, as determined by counting DAPI (Thermo Fisher Scientific) stained nucleus. Maximal respiration was calculated as the difference between FCCP and Rot/AA measurements and spare respiratory capacity was calculated as the difference between maximal and basal respiration.

Dark N., Cosson M.V., Tsansizi L.I., Owen T.J., Ferraro E., Francis A.J., Tsai S., Bouissou C., Weston A., Collinson L., Abi-Gerges N., Miller P.E., MacLeod K.T., Ehler E., Mitter R., Harding S.E., Smith J.C, & Bernardo A.S. (2023). Generation of left ventricle-like cardiomyocytes with improved structural, functional, and metabolic maturity from human pluripotent stem cells. Cell Reports Methods, 3(4), 100456.

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Measuring Mitochondrial Respiration in C2C12 Cells

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The oxygen consumption rate (OCR) in C2C12 cells was estimated using the Cell Mito Stress kit and Seahorse XFp system (Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol. Cells (1×10⁴/well) were plated, allowed to settle, and treated with fimasartan before being incubated overnight. A sensor cartridge and utility plate (cartridge+utility) containing the calibrant was incubated overnight in a CO₂-free incubator at 37℃. Oligomycin, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and antimycin A/rotenone were separately injected into each drug port in the sensor cartridge+utility plate and incubated in a CO₂-free incubator for 10 min, after which OCR was measured.

Jang Y.N., Lee Y.J., Han Y.M., Kim H.M., Seo H.S., Jeong J.H., Park S.Y, & Jung T.W. (2022). Fimasartan Ameliorates Deteriorations in Glucose Metabolism in a High Glucose State by Regulating Skeletal Muscle and Liver Cells. Yonsei Medical Journal, 63(6), 530-538.

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Metabolic Profiling of iPSC-Derived Cardiomyocytes

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Extracellular flux analyses were performed on iPSC-CMs (8×10⁴ cells) seeded on Geltrex-coated XF96 plates, with or without preincubation (24 hours) with MYK-461 (0.5 μmol/L, 1 μmol/L, or vehicle) or 2-deoxy-d-glucose (5 mmol/L or vehicle). Cellular oxygen consumption rate (OCR) and extracellular acidification rate were measured using a Cell Mito Stress Kit and a Seahorse XF96 Analyzer (Agilent Technologies) according to the manufacturer’s instructions.

Toepfer C.N., Garfinkel A.C., Venturini G., Wakimoto H., Repetti G., Alamo L., Sharma A., Agarwal R., Ewoldt J.F., Cloonan P., Letendre J., Lun M., Olivotto I., Colan S., Ashley E., Jacoby D., Michels M., Redwood C.S., Watkins H.C., Day S.M., Staples J.F., Padrón R., Chopra A., Ho C.Y., Chen C.S., Pereira A.C., Seidman J.G, & Seidman C.E. (2020). Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy. Circulation, 141(10), 828-842.

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