Xf palmitate bsa fao substrate

Manufactured by Agilent Technologies

Sourced in United States

The XF Palmitate-BSA FAO Substrate is a laboratory product designed for measuring fatty acid oxidation in cell-based assays. It provides a substrate for cellular fatty acid oxidation without additional interpretation or extrapolation.

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Lab products found in correlation

Xf cell mito stress kit, by Agilent Technologies (3 mentions) Mito stress test kit, by Agilent Technologies (2 mentions) Bovine serum albumin (bsa), by Agilent Technologies (2 mentions) Xfe wave software, by Agilent Technologies (2 mentions) Etomoxir, by Bio-Techne (1 mentions) Cyclosporin a, by Selleck Chemicals (1 mentions) Sto 609, by Bio-Techne (1 mentions) Oligomycin, by Cell Signaling Technology (1 mentions) Bezafibrate beza, by Merck Group (1 mentions) Fluo 3 am, by Beyotime (1 mentions) Bapta am, by MedChemExpress (1 mentions) Horse serum, by Thermo Fisher Scientific (1 mentions) Trypsin, by Thermo Fisher Scientific (1 mentions) Pgc1β, by Abcam (1 mentions) Uqcrc2, by Abcam (1 mentions) Ndufb8, by Abcam (1 mentions) Cox5a, by Abcam (1 mentions) Atp5a, by Abcam (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Xf96 bioanalyzer, by Agilent Technologies (1 mentions)

15 protocols using xf palmitate bsa fao substrate

Mitochondrial Function Optimization

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Etomoxir (ETO) and STO-609 was purchased from TOCRIS (Tocris Bioscience, UK). Bezafibrate (BEZA), CCCP, and H₂O₂ were obtained from Sigma (St. Louis, MO, USA). Oligomycin was purchased from Cell Signaling Technology (USA). cyclosporin A (CsA) was obtained from Selleck Chemicals (USA) and BAPTA-AM was purchased from MedChemexpress Co. (USA). The calcium-sensitive indicator, Fluo-3 AM was purchased from Beyotime Co. (China). XF Palmitate-BSA FAO Substrate was obtained from seahorse Bioscience.

Wang M.D., Wu H., Huang S., Zhang H.L., Qin C.J., Zhao L.H., Fu G.B., Zhou X., Wang X.M., Tang L., Wen W., Yang W., Tang S.H., Cao D., Guo L.N., Zeng M., Wu M.C., Yan H.X, & Wang H.Y. (2016). HBx regulates fatty acid oxidation to promote hepatocellular carcinoma survival during metabolic stress. Oncotarget, 7(6), 6711-6726.

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Mitochondrial Protein Expression Analysis

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The Eu was purchased from National Institutes for Food and Drug Control (China). Dulbecco’s modified Eagle medium (DMEM), Nutrient Mixture F-12 (DMEM/F12) media, horse serum, trypsin and penicillin/streptomycin, were obtained from Life Technologies, GibcoBRL (Rockville,MD,USA). Antibodies against CPT-1C and Tomm20 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,USA). Antibodies against ERRα and c-Myc were obtained from Cell Signaling Technology (Beverly, MA,USA) and an antibody against the HRAS, PGC-1β, NDUFB8, SDH-B, UQCRC2, ATP5A, COX5A, MCAD and PPARα were obtained from Abcam (Cambridge, MA, USA). 10058F4 were purchased from Sigma-Aldrich (St.Lois, MO, USA). XF Palmitate-BSA FAO Substrate was obtained from seahorse Bioscience (North Billerica, MA, USA).

Yan X., Zhang G., Bie F., Lv Y., Ma Y., Ma M., Wang Y., Hao X., Yuan N, & Jiang X. (2017). Eugenol inhibits oxidative phosphorylation and fatty acid oxidation via downregulation of c-Myc/PGC-1β/ERRα signaling pathway in MCF10A-ras cells. Scientific Reports, 7, 12920.

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Mitochondrial Respiration Assay with Palmitate-BSA

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The OCR was measured with a Seahorse XF96 bioanalyzer using the XF palmitate-BSA FAO substrate (Seahorse Bioscience, North Billerica, USA) and Mito Stress Test Kit (Seahorse Bioscience, North Billerica, USA) according to the manufacturer’s instructions. The OCR for oxidation of palmitate-BSA was measured in cells treated with palmitate-BSA (30 μL/well), oligomycin (1.5 μM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 2 μM), and rotenone/antimycin A (0.5 μM).

Miao Y., Zhang C., Yang L., Zeng X., Hu Y., Xue X., Dai Y, & Wei Z. (2022). The activation of PPARγ enhances Treg responses through up-regulating CD36/CPT1-mediated fatty acid oxidation and subsequent N-glycan branching of TβRII/IL-2Rα. Cell Communication and Signaling : CCS, 20, 48.

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Measuring Fatty Acid Oxidation Capacity

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Cells were seeded in an XF96 Cell Culture Microplate at 10,000 cells/well. The growth medium was replaced with substrate-limited medium (DMEM with 0.5 mM D-glucose, 1 mM L-Glutamine, 0.5 mM L-carnitine hydrochloride, and 1% FBS) to deplete endogenous substrates within the cell 24 h prior to the assay. Cells were washed with FAO Assay Medium (111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl₂, 2 mM MgSO₄, 1.2 mM NaH₂PO₄ supplemented with 2.5 mM glucose, 0.5 mM carnitine, and 5 mM HEPES and adjusted to pH 7.4) 45 min prior to the assay. FAO assay medium (135 μL/well) was added to each well and cells were equilibrated in a CO₂-free incubator for 1 h. Vehicle or Etomoxir at a final concentration of 40 μM was added to each well to inhibit CPT1 and incubated for 15 min. XF Palmitate-BSA FAO Substrate (30 μL) or BSA (Seahorse Bioscience) was added to the appropriate wells to discriminate exogenous and endogenous fatty acid oxidation. After four measurements of baseline oxygen consumption, oligomycin FCCP and antimycin A were injected. All OCR values were normalized to the total protein obtained from cells lysed by 1 M NaOH.

Hegedűs C., Juhász T., Fidrus E., Janka E.A., Juhász G., Boros G., Paragh G., Uray K., Emri G., Remenyik É, & Bai P. (2020). Cyclobutane pyrimidine dimers from UVB exposure induce a hypermetabolic state in keratinocytes via mitochondrial oxidative stress. Redox Biology, 38, 101808.

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Measuring Fatty Acid Oxidation in T Cells

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Oxidation of exogenous FFAs was measured using the XF Palmitate-BSA FAO substrate with the XF cell mito stress kit according to the manufacturer’s protocol (Seahorse Bioscience). Freshly isolated and sorted T cells (2.5 × 10⁵) were incubated for 30 min with fatty-acid oxidation assay medium (111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl₂, 2.0 mM MgSO₄, 1.2 mM Na₂HPO₄, 2.5 mM glucose, 0.5 mM carnitine and 5 mM HEPES). When required, cells were pre-treated with etomoxir (40 μM) for 15 min. Afterwards, BSA (34 μM) or palmitate-BSA (200 μM palmitate conjugated with 34 μM BSA) was added to the medium, and the OCR was measured under basal conditions and in response to 1 μM oligomycin, 1.5 μM fluorocarbonyl cyanide phenylhydrazone (FCCP), and 100 nM rotenone + 1 μM antimycin A. Results were normalized to data from control cells in the presence of BSA.

Pan Y., Tian T., Park C.O., Lofftus S.Y., Mei S., Liu X., Luo C., O’Malley J.T., Gehad A., Teague J.E., Divito S.J., Fuhlbrigge R., Puigserver P., Krueger J.G., Hotamisligil G.S., Clark R.A, & Kupper T.S. (2017). Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism. Nature, 543(7644), 252-256.

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Measuring Cellular Fatty Acid Oxidation

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To measure fatty acid oxidation (FAO) cell medium was changed one day before the assay to Substrate Limited Medium (DMEM (A14430–01-Gibco) supplemented with 0.5 mM glucose (Sigma), 1 mM glutamax (Gibco), 0.5 mM L-Carnitine (Sigma) and 1% FBS (Sigma)). At the day of experiment cells were placed in FAO assay medium (111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl₂, 2 mM MgSO₄, 1.2 mM NaH₂PO₄) supplemented with 2.5 mM glucose, 0.5 mM carnitine, and 5 mM HEPES, pH 7.4 at 37 °C. FAO assay was performed using XF Palmitate-BSA FAO Substrate (Seahorse Bioscience) according to manufacturer’s recommendations. Mitochondrial function was analyzed as described above with 3–7 biological replicates per patient and data were pooled to give average values for each condition.

Fontes-Oliveira C.C., Steinz M., Schneiderat P., Mulder H, & Durbeej M. (2017). Bioenergetic Impairment in Congenital Muscular Dystrophy Type 1A and Leigh Syndrome Muscle Cells. Scientific Reports, 7, 45272.

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Palmitate-Induced Metabolic Dysfunction

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XF Palmitate-BSA FAO Substrate (Seahorse Bioscience) was dissolved in an FCS-free medium, then the final concentration of palmitate in the medium was adjusted to 0.3 mmol/L. At 20 min before palmitate stimulation, 10 µmol/L astaxanthin, 1 mmol/L NAC, 10 μmol/L SP600125, or 10 μmol/L LY294002 was added in the culture medium. At 24h after the exposure to palmitate, the cell lysates and medium were subjected to various experiments.
In other experimental series, MIN6 cells were pretreated with astaxanthin, and then after 20 min, 0.3 mmol/L H₂O₂ was added into the culture medium. At 24 h after H₂O₂ administration, MCP-1 and VEGF₁₂₀ secretion were analyzed by immunoblotting.

Kitahara A., Takahashi K., Morita N., Murashima T., Onuma H., Sumitani Y., Tanaka T., Kondo T., Hosaka T, & Ishida H. (2017). The Novel Mechanisms Concerning the Inhibitions of Palmitate-Induced Proinflammatory Factor Releases and Endogenous Cellular Stress with Astaxanthin on MIN6 β-Cells. Marine Drugs, 15(6), 185.

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Fatty Acid Oxidation in Naive CD4 T Cells

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Oxidation of exogenous free fatty acids was assayed utilizing the XF Palmitate-BSA FAO substrate with the XF cell mito stress kit according to the manufacturer’s protocol (Seahorse Bioscience). Negatively selected naive CD4 T cells (4×10⁵ cells) were incubated for 30 minutes with fatty acid oxidation assay medium (111mM NaCl, 4.7mM KCl, 1.25mM CaCl₂, 2.0mM MgSO₄, 1.2mM Na₂HPO₄, 2.5mM glucose, 0.5mM carnitine, and 5mM HEPES). To block fatty acid oxidation, naive CD4 T cells were pre-treated with 40μM etomoxir for 15 minutes at 37°C. Afterwards, 34μM BSA or palmitate-BSA (200μM palmitate conjugated with 34μM BSA) was added to the medium and the OCR was assayed under basal conditions and in response to 1μM oligomycin, 1.5μM fluorocarbonyl cyanide phenylhydrazone (FCCP), and 100nM rotenone + 1μM antimycin A.

Gaddis D.E., Padgett L.E., Wu R., Nguyen A., McSkimming C., Dinh H.Q., Araujo D.J., Taylor A.M., McNamara C.A, & Hedrick C.C. (2021). Atherosclerosis impairs naive CD4 T cell responses via disruption of glycolysis. Arteriosclerosis, thrombosis, and vascular biology, 41(9), 2387-2398.

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Fatty Acid Oxidation Substrate Assay

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XF Palmitate-BSA FAO Substrate was purchased from Seahorse Bioscience (North Billerica, MA, USA), and astaxanthin was obtained from Fuji Chemical Industry Co., Ltd. (Toyama, Japan). SP600125 and LY294002 were purchased from A.G. Scientific, Inc. (San Diego, CA, USA). Antibodies against CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), Akt1/2/3 (phospho Tyr315/316/312) and phosphorylated c-Jun NH₂-terminal protein kinase (JNK) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA)., and antibody against phosphorylated Akt1/2/3 from Assay Designs (Ann Arbor, MI, USA). Antibodies against MCP-1, VEGF₁₂₀, IL-10, JNK, and anti-glucose-regulated protein (GRP78)/binding immunoglobulin protein (Bip) were obtained from R&D Systems (Minneapolis, MN, USA), and N-acetyl-cysteine (NAC), hydrogen peroxide (H₂O₂), and the antibody against β-Actin from Sigma-Aldrich (St. Louis, MO, USA).

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Evaluating Mitochondrial Function and Fatty Acid Oxidation in BM-MDSCs

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The Seahorse XF Cell Mito Stress Test Kit (Cat# 103015-100, Seahorse Bioscience) was used ccording to the manufacturer’s protocol to determine the mitochondrial function of BM-MDSCs by measuring the OCR with an XF96 analyzer (Seahorse Bioscience). About 2 × 10⁵ cells were seeded per well in a XF96 cell culture microplate and compounds were injected during the assay at the following final concentrations: 1.0 μM oligomycin, 1.5 μM FCCP, 0.5 μM rotenone, 0.5 μM antimycin A, and 100 μM etomoxir. The XF-palmitate-BSA FAO substrate (Cat# 102720-100, Seahorse Bioscience) was used to evaluate the oxidation of exogenously added fatty acids and the proportion of respiration supported by exogenous fatty acids according to the manufacturer’s protocol (Seahorse Bioscience, #102720-100). The BM-MDSCs were inoculated with either palmitate-BSA (200 µM palmitate conjugated with 34 µM BSA) or BSA (34 µM) (Seahorse Bioscience) and the OCR was analyzed as described above.

Xin G., Chen Y., Topchyan P., Kasmani M.Y., Burns R., Volberding P.J., Wu X., Cohn A., Chen Y., Lin C.W., Ho P.C., Silverstein R., Dwinell M.B, & Cui W. (2021). Targeting PIM1-Mediated Metabolism in Myeloid Suppressor Cells to Treat Cancer. Cancer immunology research, 9(4), 454-469.

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