Rot aa

Manufactured by Agilent Technologies

Sourced in United States

The Rot/AA is a laboratory equipment designed for rotary evaporation and atomic absorption spectroscopy. It provides a controlled environment for solvent evaporation and sample preparation, as well as the ability to perform atomic absorption analysis.

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5 protocols using rot aa

Assessing Hepatocyte Mitochondrial Function

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Hepatocytes were plated in a collagen-coated (Sigma-Aldrich C3867) seahorse XF96 cell culture microplates at 5000 cells/well in 80 µL in Buffer C and incubated at 37 °C for 6 h. After 6 h media was changed, and hepatocytes were cultured overnight before any experimental procedure. Mitochondrial measurements and FAO were performed using the Seahorse XF Cell Mito Stress Test (Agilent 103015-100) according to manufacturer’s instructions. Briefly, primary hepatocytes were plated as indicated above and the following morning regular media was replaced for Seahorse XF DMEM medium (Agilent 103575-100) supplemented with 10 mM glucose (Agilent 103577-100), 1 mM Sodium pyruvate (Agilent 103578-100) and 2 mM glutamine (Agilent 103579-100). Oxygen consumption Rate (OCR) was measured following the sequential addition of 40 µM of Etomoxir (MedChemTronica HY-502002; port A), 1.5 µM of oligomycin (port B), 0.3 µM of carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP)(port C) and 0.5 µM of Antimycin/Rotenone (Rot/AA) (port C). The oligomycin, FCCP and the Rot/AA were purchase from Agilent (103015-100). Oxygen consumption rate (OCR) was normalized to protein concentration using BCA Protein Assay (Thermo Scientific™ 23222). All parameters were represented as normalize OCR.

de la Calle Arregui C., Plata-Gómez A.B., Deleyto-Seldas N., García F., Ortega-Molina A., Abril-Garrido J., Rodriguez E., Nemazanyy I., Tribouillard L., de Martino A., Caleiras E., Campos-Olivas R., Mulero F., Laplante M., Muñoz J., Pende M., Sabio G., Sabatini D.M, & Efeyan A. (2021). Limited survival and impaired hepatic fasting metabolism in mice with constitutive Rag GTPase signaling. Nature Communications, 12, 3660.

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Extracellular Acidification of Mouse Urothelial Cells

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After the urothelial cells were scrapped off from freshly dissected mouse bladders, they were digested in a Liberase solution (Roche Diagnostics, Florham Park, NJ) at 37°C for 20 min. The reaction was stopped by the addition 2% fetal bovine serum containing 0.2 M EDTA. The urothelial cell suspension was then transferred into 24-well plates pre-coated with poly-L-lysine (Seahorse Bioscience, Santa Clara, CA) and incubated with XF Base DMEM medium (Agilent, Santa Clara, CA) at 37 °C in 5% CO₂. For determination of extracellular acidification rate (ECAR), the following were added: 10 mM glucose, 0.5 μM Rot/AA (rotenone/antimycin A) and 50 mM 2-DG (2-deoxy-glucose) (Agilent, Santa Clara, CA, USA) at the time points indicated in Fig. 8 into XFe24 extracellular flux analyzer (Agilent, Santa Clara, CA).

Xia Y., Wang X., Liu Y., Shapiro E., Lepor H., Tang M.S., Sun T.T, & Wu X.R. (2022). PKM2 is essential for bladder cancer growth and maintenance. Cancer research, 82(4), 571-585.

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Metabolic Profiling of Muropeptide Effects

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OCR measurements were performed with a Seahorse XF-24 analyzer (Seahorse Bioscience). Cells were seeded in a Seahorse XF-24 cell culture microplate [1.7 ×10⁵ caco-2 cells per well in 250 μL DMEM media (Gibco, 11995040) supplemented with 10% heat-in-activated FBS and 1% non-essential amino acid, or 1.0 ×10⁴ fibroblast cells per well in 200 μL EMEM media supplemented with 15% FBS] and then incubated at 37°C and 5% CO₂ overnight. After overnight recovery, the cells were incubated with muropeptide solution or lysozyme solution (mock) for 24 h. Cells were washed in Seahorse XF DMEM media (Agilent, 30119005, supplemented with 1mM pyruvate, 10 mM glucose and 2 mM glutamine) and incubated at 37°C with no CO₂ for 1 h before measurement. Cells were washed again with Seahorse XF DMEM media just before measuring. OCR was measured and analyzed by adding 2.5 μM oligomycin, 1μM FCCP and 1.25 μM ROT/AA (Agilent, 103015–100) sequentially following the manufacture’s instruction. After OCR measurements, cells from each well were lysed in lysis buffer (100 mM NaCl, 0.5mM EDTA, 0.5% NP-40 and a cocktail of protease inhibitors in 10 mM Tris-HCl, pH7.4) and total proteins were quantified by using the BCA analysis.

Tian D., Cui M, & Han M. (2024). Bacterial muropeptides promote OXPHOS and suppress mitochondrial stress in mammals. Cell reports, 43(4), 114067.

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Real-Time ATP Measurement in Cells

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Cells were plated at the optimal densities in the Seahorse XF 8-well plates 48/72 h before the measurement, subjected to the FF treatment for 24/48 h and incubated in the Seahorse XF Assay Media at 37 °C for 1 h without CO₂, immediately before starting the Real-Time ATP Rate Assay (Seahorse Bioscience; North Billerica, MA, USA, 1 μM Oligo; 1 μM/0.5 μM Rot/AA). OCR and ECAR measurements were performed with the Seahorse Analyzer XF HS Mini/XFp software and normalized to cell numbers.

Warzecha K.W., Pudełek M., Catapano J., Madeja Z, & Czyż J. (2022). Long-Term Fenofibrate Treatment Stimulates the Phenotypic Microevolution of Prostate Cancer Cells In Vitro. Pharmaceuticals, 15(11), 1320.

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Mitochondrial Respiration Assay Protocol

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Cells were plated at optimal densities in Seahorse XF 24-well plates one or two days prior to the measurement. Cells were incubated with Seahorse XF Assay Media at 37 °C for 1 h without CO₂ for basal OCR and with MAS buffer (Mannitol and sucrose buffer: 70 mM sucrose, 220 mM Mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, and 1 mM EGTA in diH2O. pH 7.2 using 0.1 M KOH) to measure complex activity just before running the assay. Substrate concentrations were 1μM for Oligo and FCCP, 1μM/0.5μM for Rot/AA, and 5 mM for succinate, all the substrates were purchased from Seahorse Bioscience. Reagents for complex activity such as Saponin 100 μg/ml, Pyruvate 10 mM, Malate 2 mM, ADP 50 μM and NADH 10 mM were purchased from Sigma. OCR measurements were obtained using the Seahorse XFe24 Analyzer, and normalized to protein concentration (µg/µL).

Lee B.W., Chuah Y.H., Yoon J., Grinchuk O.V., Liang Y., Hirpara J.L., Shen Y., Wang L.C., Lim Y.T., Zhao T., Sobota R.M., Yeo T.T., Wong A.L., Teo K., Nga V.D., Tan B.W., Suda T., Toh T.B., Pervaiz S., Lin Z, & Ong D.S. (2024). METTL8 links mt-tRNA m3C modification to the HIF1α/RTK/Akt axis to sustain GBM stemness and tumorigenicity. Cell Death & Disease, 15(5), 338.

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