Sodium pyruvate

Manufactured by Agilent Technologies

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Sodium pyruvate is a chemical compound that is commonly used in cell culture media as an energy source for cells. It is a salt of pyruvic acid, which is a key intermediate in cellular metabolism. Sodium pyruvate is a white, crystalline powder that is soluble in water and is typically used in cell culture applications to support cell growth and proliferation.

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12 protocols using sodium pyruvate

Mitochondrial Respiration Profiling in Cells

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OCR was assessed using the Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies). Cells were seeded quadruplicately in cell culture microplates (Agilent Technologies, Santa Clara, CA, USA) and treated with control or HP for 24 h. One hour prior to the assay, the culture medium was replaced by base medium (Agilent Technologies) supplemented with 1 mM sodium pyruvate, 2 mM glutamine, and 10 mM glucose (Agilent Technologies), and then cells were incubated at 37 °C without CO₂. After three basal respiration measurements without additives, the ATP synthase inhibitor (1 μM oligomycin) was added, followed by subsequent mitochondrial uncoupler (1 μM FCCP), and complex I and III inhibitors (1 μM rotenone and 1 μM antimycin A), respectively. Three separate measurements were recorded after the reagents (all bought from Agilent Technologies) were injected.

Huang Y., Wang S., Zhou J., Liu Y., Du C., Yang K., Bi X., Liu M., Han W., Wang K., Xiong J., Wang S., Wang Y., Nie L., Liu C., Zhang D., Gu J., Zeng C, & Zhao J. (2020). IRF1-mediated downregulation of PGC1α contributes to cardiorenal syndrome type 4. Nature Communications, 11, 4664.

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Mitochondrial Stress Test of Activated CD8+ T-cells

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The OCR and ECAR of in vitro-activated CD8⁺ T-cells were measured in XF RPMI media containing 25 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate (Agilent, Lexington, MA, USA). Cells were then plated onto XF8 cell culture microplates (1.5 × 10⁵ cells per well) coated with poly-D-lysine (Sigma-Aldrich) to facilitate T-cell attachment. A mitochondrial stress test was performed by measuring OCR (pmol min-1) at the basal level and after sequential injection of oligomycin (1.5 μM), FCCP (2.5 μM), and rotenone/antimycin A (0.5 μM) (Agilent, CA, USA), and run on an Agilent Seahorse XFp analyzer (Seahorse Bioscience, Agilent, CA, USA). The following assay conditions were used for the experiments with the Seahorse system: 3 min mixture; 0 min wait; and 3 min measurement.

Ara A., Xu A., Ahmed K.A., Leary S.C., Islam M.F., Wu Z., Chibbar R, & Xiang J. (2021). The Energy Sensor AMPKα1 Is Critical in Rapamycin-Inhibition of mTORC1-S6K-Induced T-cell Memory. International Journal of Molecular Sciences, 23(1), 37.

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Mitochondrial Respiration and Hepatic Lipids

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Oxygen consumption rate (OCR) of Hepa 1-6 cells with siRNA-mediated gene suppression was measured using the Seahorse Flux Analyzer XF24 (Agilent). The cells were transfected with 120 nM of siASIrs2 or control siRNA in DMEM (High glucose) medium supplemented with penicillin and streptomycin and 10% fetal bovine serum. 48 hours later, the medium was changed to XF Base Medium (Agilent) supplemented with 25 mM of glucose, 4 mM of glutamine and 1mM of sodium pyruvate (Agilent), and OCR measurement was conducted with subsequent stimulation by 1.5 mM oligomycin, 0.5 mM fluoro-carbonyl cyanide phenylhydrazone (FCCP) and 0.5 mM Rotenone/Antimycin A (Seahorse XF Cell Mito Stress Test Kit, Agilent) according to the manufacture's protocol.
Triglyceride measurement of liver Around 100 mg of freshly frozen liver pieces were completely digested with 1700 ml of ethanolic KOH (EtOH:30% KOH = 2:1) for overnight at 55 C with vigorous shaking. 200 ml of the lysate was mixed with 215 ml of 1M MgCl2, and after incubation on ice for 5 minutes, was centrifuged. The supernatant was subjected to the triglyceride measurement using Triglyceride-E Test kit (WAKO).

Matsushita M., Awazawa M., Kobayashi N., Ikushima Y.M., Soeda K., Tamura-Nakano M., Muratani M., Kobayashi K., Blüher M., Brüning J.C, & Ueki K. (2022). An antisense transcript transcribed from Irs2 locus contributes to the pathogenesis of hepatic steatosis in insulin resistance. Cell chemical biology, 29(4).

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Measuring Cellular Metabolism in HDFs

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The glycolysis rate and the oxygen consumption rate (OCR) in HDFs were measured with a Seahorse XFe24 analyzer (Agilent: Seahorse XFe24 analyzer, Santa Clara, CA, USA) and Wave Software (Agilent: wave controller, https://www.agilent.com/en/product/cell-analysis/real-time-cell-metabolic-analysis/xf-software/seahorse-wave-desktop-software-740897, accessed on 5 March 2022). HDFs were seeded the previous day to reach 95% confluency before measuring. The glycolytic stress test measured the extracellular acidification rate (ECAR) following the manufacturer’s instructions. The OCR was measured using the Mito Stress test. DMEM media supplemented with 2 mM of L-glutamine (Thermofisher, Waltham, MA, USA) was used for the glycolytic stress test. A total of 10 mM of glucose (Agilent), 1 μM of oligomycin (Agilent), and 50 mM of 2-Deoxy-D-glucose (2-DG) (Agilent) were injected during the assay. For the MitoStress test, DMEM media supplemented with 10 mM of glucose, 1 mM of sodium pyruvate (Agilent), and 2 mM of L-glutamine were used. In total, 1.5 μM of oligomycin, 2 μM of FCCP (Agilent), and 0.5 μM of rotenone AA (Agilent) were injected during the assay.

Heo J.W., Lee H.E., Lee J., Choi L.S., Shin J., Mun J.Y., Park H.S., Park S.C, & Nam C.H. (2024). Vutiglabridin Alleviates Cellular Senescence with Metabolic Regulation and Circadian Clock in Human Dermal Fibroblasts. Antioxidants, 13(1), 109.

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Measuring Cellular Respiration in EPSCs, Fusion Hybrids, and NSCs

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A Seahorse XFp analyzer was used to measure the oxygen consumption rate (OCR). Overall, 6 × 10⁴ cells of EPSCs, fusion hybrid cells, and 8 × 10⁴ cells of NSCs were cultured for 16 h after being seeded into a cell culture plate pre-coated with diluted Matrigel (Corning, NY, USA, 356234). For analysis, the medium was changed to XFp base media supplemented with D-glucose (Agilent, Santa Clara, CA, USA, 103577-100), sodium pyruvate (Agilent, 103578-100), and L-glutamine (Agilent, 103579-100). The OCR was measured using a Seahorse XFp analyzer (Seahorse Bioscience, Billerica, MA, USA). Measurement values were obtained after injection of oligomycin (1.5 µM), FCCP (0.1 µM), and rotenone/antimycin A (0.5 µM) (Agilent). The analysis was performed according to the manufacturer’s instructions.

Song J.H., Choi J., Hong Y.J., La H., Hong T.K., Hong K, & Do J.T. (2022). Developmental Potency and Metabolic Traits of Extended Pluripotency Are Faithfully Transferred to Somatic Cells via Cell Fusion-Induced Reprogramming. Cells, 11(20), 3266.

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Quantifying Cellular Respiration with Seahorse

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Real-time measurements of oxygen consumption rates (OCR) were performed using a Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies). For ISO-induced assays, the cell culture medium was changed 1 h before the first measurement to XF assay medium-modified DMEM (Agilent Technologies) supplemented with 5 mM of glucose and 2 mM of l-glutamine and adjusted to pH 7.4. The OCR were measured under basal conditions and after injection of 10 μM of isoproterenol, 1 μM of FCCP, and 1 μM of rotenone combined with 1 μM of antimycin A. The area under the curve (AUC) was calculated from the time of the isoproterenol injection (ISO-induced OCR). For Mito stress tests, DMEM (Agilent Technologies) was supplemented with 10 mM of glucose, 2 mM of glutamine, and 1 mM of sodium pyruvate (Agilent Technologies). The OCR were measured under basal conditions and following an injection of oligomycin (5 μM), FCCP (1 μM), and rotenone/antimycin A (1 μM) (Agilent Technologies). Calculations were performed using the Seahorse report generator.

Rabiee A., Plucińska K., Isidor M.S., Brown E.L., Tozzi M., Sidoli S., Petersen P.S., Agueda-Oyarzabal M., Torsetnes S.B., Chehabi G.N., Lundh M., Altıntaş A., Barrès R., Jensen O.N., Gerhart-Hines Z, & Emanuelli B. (2020). White adipose remodeling during browning in mice involves YBX1 to drive thermogenic commitment. Molecular Metabolism, 44, 101137.

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Real-Time Cellular ATP Kinetics Assay

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The Agilent Seahorse XF Real-Time ATP Rate Assay was conducted following the manufacturer’s recommendations using the ATP rate assay kit (Agilent, Catalog # 103592-100). BMDMs were isolated and plated, then treated with LPS as described above. The Seahorse media for the ATP rate assay was prepared according to the manufacturer’s instructions the day before the assay and supplemented with 15 mM glucose (Sigma), 4 mM glutamine (Gemini) and 1 mM sodium pyruvate (Agilent). The assay was conducted according to the manufacturer’s protocol. The final concentrations of the injection compounds used in this assay were oligomycin (1 µM) and rotenone/antimycin A (0.5 µM). At the end of the assays, the cells were collected in RIPA buffer for protein estimation. The data were collected and analyzed using Wave software (Agilent).

Shosha E., Shahror R.A., Morris C.A., Xu Z., Lucas R., McGee-Lawrence M.E., Rusch N.J., Caldwell R.B, & Fouda A.Y. (2023). The arginase 1/ornithine decarboxylase pathway suppresses HDAC3 to ameliorate the myeloid cell inflammatory response: implications for retinal ischemic injury. Cell Death & Disease, 14(9), 621.

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Seahorse XFp Metabolic Analysis of Neuronal Cells

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After 24 h of transfection, cells were seeded on XFp Miniplate (Agilent) at a density of 20,000 cells/80 μL for H4-APPswe and 40,000 cells/80 μL for SH-SY5Y. After 24 h, media was changed to 180 μL of Seahorse XF DMEM (Agilent) supplemented with 10 mM glucose (Agilent), 1 mM sodium pyruvate (Agilent) and 1x Glutamax (Gibco). OCR was analyzed using Seahorse XFp analyzer (Agilent) with sequential addition of 1.5 μM Oligomycin, carbonyl cyaide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 2 μM for H4-APPswe and 0.5 μM for SH-SY5Y) and 0.5 μM rotenone/antimycin A in Seahorse XFp Cell Mito Stress Test Kit (Agilent). The data were analyzed using WAVE software (Agilent) and normalized by cell viability.

Kim S.H., Choi K.Y., Park Y., McLean C., Park J., Lee J.H., Lee K.H., Kim B.C., Huh Y.H., Lee K.H, & Song W.K. (2021). Enhanced Expression of microRNA-1273g-3p Contributes to Alzheimer’s Disease Pathogenesis by Regulating the Expression of Mitochondrial Genes. Cells, 10(10), 2697.

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Seahorse XF Metabolic Profiling of Primary B Cells

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Oxygen consumption rate and extracellular acidification rate were measured with the Seahorse XF96 metabolite analyzer using the Seahorse XF Cell Mito Stress Test kit (Agilent). Primary B cells were isolated, counted, and plated at the indicated cell densities directly on Seahorse cell culture plates coated with poly-l-lysine (Sigma-Aldrich). Before measurement, cells were incubated for a maximum of 1 h in Seahorse XF DMEM medium pH 7.4 (Agilent) supplemented with 10 mM glucose (Agilent), 1 mM sodium pyruvate (Agilent), and 2 mM l-glutamine (Agilent).

Delgado-Benito V., Berruezo-Llacuna M., Altwasser R., Winkler W., Sundaravinayagam D., Balasubramanian S., Caganova M., Graf R., Rahjouei A., Henke M.T., Driesner M., Keller L., Prigione A., Janz M., Akalin A, & Di Virgilio M. (2020). PDGFA-associated protein 1 protects mature B lymphocytes from stress-induced cell death and promotes antibody gene diversification. The Journal of Experimental Medicine, 217(10), e20200137.

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Metabolic Effects of Naringenin Compound

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Hyclone™ Dulbecco’s Modified Eagle Medium/Ham’s F12 (DMEM/F12 1:1) and Gibco™ fetal bovine serum (FBS) were purchased from ThermoFisher Scientific (Waltham, MA), Rosiglitazone was purchased from AK Scientific (Union City, CA). All other reagents used in the medium were purchased from Sigma-Aldrich (St. Louis, MO). Naringenin (purity ≥ 98%) was purchased from Cayman Chemicals (Ann Arbor, MI). Protease and phosphatase were purchased from Cell Signaling Technology (Danvers, MA), RIPA buffer from Sigma, TGX protein gels from BIO-RAD (Hercules, CA). Seahorse Assay Medium and sodium pyruvate were purchased from Agilent (Santa Clara, CA). carbonyl cyanide-4-(trifluoromethoxy) phenyhydrazone (FCCP), Oligomycin, and Antimycin A were purchased from Sigma-Aldrich.

Rebello C., Greenway F.L., Lau F.H., Lin Y., Stephens J.M., Johnson W.D, & Coulter A.A. (2018). Naringenin Promotes Thermogenic Gene Expression in Human White Adipose Tissue. Obesity (Silver Spring, Md.), 27(1), 103-111.

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