Agilent seahorse mito stress test kit

Manufactured by Agilent Technologies

Sourced in United States

The Agilent Seahorse Mito Stress Test kit is a laboratory equipment product designed to measure the mitochondrial function of cells. It provides real-time analysis of key parameters such as oxygen consumption rate and extracellular acidification rate, which reflect the cellular energy metabolism.

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Lab products found in correlation

Seahorse xfe96, by Agilent Technologies (2 mentions) Xfp analyzer, by Agilent Technologies (2 mentions) Xf mito stress test kit, by Agilent Technologies (2 mentions) Xf 96 well analyzer, by Agilent Technologies (1 mentions) Poly da dt, by InvivoGen (1 mentions) Lipopolysaccharides (lps), by InvivoGen (1 mentions)

4 protocols using agilent seahorse mito stress test kit

Seahorse Analyzer Mitochondrial Respiration

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OCR was determined using a seahorse XFe96 or XFp analyzer (Agilent Technologies, Santa Clara, CA, USA) accompanied by an Agilent Seahorse Mito Stress Test kit (Agilent Technologies) according to the manufacturer’s instructions. Key parameters of mitochondrial respiration were analyzed in cells treated with 1 μM oligomycin, 1 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and a mixture of 0.5 μM antimycin A/rotenone. At the end of the Seahorse assay, a protein assay was performed to normalize the OCR measurements. OCR values were normalized for the amount of cellular protein in each well.

Kim S.R., Park J.W., Lee B.H., Lim K.M, & Chang T.S. (2023). Peroxiredoxin V Protects against UVB-Induced Damage of Keratinocytes. Antioxidants, 12(7), 1435.

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Seahorse XF Assay for Inflammasome Activation

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Seahorse XF mito stress test kits and cartridges were prepared per Agilent’s protocols and analyzed on an Agilent Seahorse XF 96-well analyzer using WAVE software post analysis. The day before the assay BMDMs were seeded at 5×10⁴ cells/well overnight. For inflammasome activation on the day of the assay cells were treated with 10 ng/mL LPS (Invivogen), followed by 1 μg/mL poly dA:dT (Invivogen) 3 hrs later. After 1 hr of AIM2 activation cells were processed per manufacturer’s directions and analyzed using the Agilent Seahorse Mito Stress Test kit (Agilent). For overnight stimulation with LPS, cells were treated 2 hrs after plating with 10 ng/mL LPS and incubated overnight at 37 °C.

Weindel C.G., Martinez E.L., Zhao X., Mabry C.J., Bell S.L., Vail K.J., Coleman A.K., VanPortfliet J.J., Zhao B., Wagner A.R., Azam S., Scott H.M., Li P., West A.P., Karpac J., Patrick K.L, & Watson R.O. (2022). Mitochondrial ROS promotes susceptibility to infection via gasdermin D-mediated necroptosis. Cell, 185(17), 3214-3231.e23.

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Seahorse XF Mito Stress Test in RAW MΦ

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Seahorse XF mito stress test kits and cartridges were prepared per Agilent’s protocols and analyzed on an Agilent Seahorse XF 96-well analyzer. The day before the assay Srsf6 knockdown RAW MΦ were seeded at 5×10⁴ cells per well and rested overnight. Cells were processed per manufacturer’s directions and analyzed using the Agilent Seahorse Mito Stress Test kit (Agilent, 103015–100). Normalization was performed based on absorbance (Abs 562) of total protein concentration measured using a bicinchoninic acid assay (BCA) (Thermo Fisher Scientific, 23225). WAVE software was used for post-acquisition analysis.

Wagner A.R., Weindel C.G., West K.O., Scott H.M., Watson R.O, & Patrick K.L. (2022). SRSF6 balances mitochondrial-driven innate immune outcomes through alternative splicing of BAX. eLife, 11, e82244.

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Measurement of Cellular Mitochondrial Respiration

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Cells were cultured in Seahorse XFp plates at a density of 4 × 10⁴ cells/well in DMEM containing 10% FBS and 1% antibiotics–antimycotics. OCR was determined using a seahorse XFe96 or XFp analyzer (Agilent Technologies, Santa Clara, CA, USA) accompanied by an Agilent Seahorse Mito Stress Test kit (Agilent Technologies) according to the manufacturer’s instructions. Key parameters of mitochondrial respiration were analyzed in cells treated with 1 μM oligomycin, 1 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and a mixture of 0.5 μM antimycin A/rotenone. At the end of the Seahorse assay, a protein assay was performed to normalize the OCR measurements. OCR values were normalized for the amount of cellular protein in each well.

Kim S.R., Park J.W., Choi Y.J., Sonn S.K., Oh G.T., Lee B.H, & Chang T.S. (2023). Mitochondrial H2O2 Is a Central Mediator of Diclofenac-Induced Hepatocellular Injury. Antioxidants, 13(1), 17.

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