Sodium pyruvate

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Sodium pyruvate is a chemical compound that serves as a key component in various cell culture media and biochemical assays. It functions as an energy substrate and is essential for maintaining the viability and growth of cells in vitro.

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13 protocols using sodium pyruvate

Induced Differentiation of Fetal Germ Stem Cells

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Induction of FGSC differentiation was performed as described in a previous report.¹⁰ Briefly, STO feeder cells (derived from mouse SIM embryonic fibroblasts, strain SIM, ATCC, Manassas, VA, USA) were removed from FGSCs cultured by differential adherence. FGSCs were harvested and seeded on freshly mitotically inactivated granulosa cells (GCs) in differentiation medium containing MEMα supplemented with 5% fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, USA), 10 ng/ml bFGF, 10 ng/ml bone morphogenic protein (BMP4; PeproTech, NJ, USA), 0.1 μmol/L retinoic acid (RA; Sigma‐Aldrich, St. Louis, MO, USA), 1 mM nonessential amino acids (NEAA; Invitrogen Life Sciences), 2 mM L‐glutamine (Sigma‐Aldrich), 1 mM sodium pyruvate (Amresco, Radnor, PA, USA) and 0.1 mM β‐mercaptoethanol (Sigma‐Aldrich). The medium was changed every 2 days.

Zhang Y., Tian G.G., Wang X., Hou C., Hu X, & Wu J. (2022). Retinoic acid induced meiosis initiation in female germline stem cells by remodelling three‐dimensional chromatin structure. Cell Proliferation, 55(7), e13242.

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Cell Culture and Antibody Reagents

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Mardin-Darby canine kidney (MDCK) cells, human embryonic kidney (HEK) 293T cells, and human lung epithelial carcinoma A549 cells were cultured in this study with DMEM medium (Invitrogen, Carlsbad, CA), 10% fetal bovine serum (HyClone, Logan, UT), 1 mM sodium pyruvate (Amresco, Solon, OH), and 1% antibiotic-antimycotic solutions (Invitrogen) at 37°C in an incubator with constant 5% CO₂. Transfection reagent TransIT-LT1 was purchased from Mirus Bio (Madison, WI). Primary antibodies for cytochrome c (sc-7159), HA tag (sc-805), β-actin (sc-47778), and HAX-1 (sc-166845) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and Flag-tag M2 monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO). Anti-cleaved caspase-9 (number 9501) antibody, anti-cleaved caspase-3 (number 9664) antibody, and horseradish peroxidase (HRP) or alkaline phosphatase- (AP-) conjugated anti-rabbit IgG were obtained from Cell Signaling Technology (Danvers, MA). HRP-conjugated anti-mouse IgG was purchased from Sunshine Biotechnology (Nanjing, China).

Li X., Qu B., He G., Cardona C.J., Song Y, & Xing Z. (2018). Critical Role of HAX-1 in Promoting Avian Influenza Virus Replication in Lung Epithelial Cells. Mediators of Inflammation, 2018, 3586132.

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Detailed Culture Protocol for Mouse SSCs

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The cells used in this study were an SSC line that we had established previously 13 (link), 20 . The SSC culture system employed a mitotically inactivated STO (SIM mouse embryo-derived thioguanine and ouabainresistant feeder) feeder layer. STO cells were cultured in DMEM supplemented with fetal bovine serum (FBS, Life Technologies), 2 mM glutamine (Amresco), 100 µg/ml penicillin (Amresco), and 100 µg/ml streptomycin (Amresco) (STO culture medium). The culture medium for SSCs was alpha-minimum essential medium supplemented with 1 mM sodium pyruvate (Amresco), 10% fetal bovine serum (FBS, Life Technologies), 10 ng/ml mouse glial cell line‐derived neurotrophic factor (GDNF) (PeproTech), 1 mM non-essential amino acids (Invitrogen), 2 mM L-glutamine (Amresco), 0.1 mM β-mercaptoethanol (Biotech), 10 ng/ml leukemia inhibitory factor (Santa Cruz Biotechnology, CA, USA), 20 µg/ml transferrin (Sigma), 60 µM putrescine, 20 ng/ml mouse epidermal growth factor (PeproTech), 5 µg/ml insulin, 30 mg/l penicillin (Amresco), 75 mg/l streptomycin (Amresco) (SSC medium), and 10 ng/ml human basic fibroblast growth factor (PeproTech). The medium was changed every 2-3 days, and cells were subcultured at 1:2 or 1:3 ratios by enzymatic digestion every 5-7 days. The cells were maintained at 37°C in 5% CO₂20 .

Wang Y., Li X., Gong X., Zhao Y, & Wu J. (2019). MicroRNA-322 Regulates Self-renewal of Mouse Spermatogonial Stem Cells through Rassf8. International Journal of Biological Sciences, 15(4), 857-869.

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Isolation and Culture of Mouse Spermatogonial Stem Cells

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We isolated and purified SSCs from neonatal mouse testes (6 days old, C57BL/6) following a previously described protocol [48 (link)]. We cultured SSCs on STO feeder cells in MEMα basal medium (Invitrogen) supplemented with 10% FBS (Life Technologies), 1 mM sodium pyruvate (Amresco), 2 mM L-glutamine (Amresco), 50 μM β-mercaptoethanol (Biotech), 1 mM NEAA (Invitrogen), 20 ng/ml mouse EGF (PeproTech), 10 ng/ml human bFGF (PeproTech), 10 ng/ml mouse GDNF (PeproTech), 10 ng/ml ESGRO (Santa Cruz Biotechnology), and 100 μg/ml transferrin (Sigma). Spermatogonial stem cells were identified by immunofluorescence staining and RT-PCR (Additional file 1: Figure S2).

Hu X., Wang H., Tian G.G., Hou C., Xu B., Zhao X., Zhao Y., Fang Q., Li X., He L., Chen X., Li S, & Wu J. (2022). Offspring production of haploid spermatid-like cells derived from mouse female germline stem cells with chromatin condensation. Cell & Bioscience, 12, 5.

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Diverse Reagents for Microbial Cultivation

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Zobell marine broth 2216 (HiMedia lab), sodium pyruvate (Amresco), nutrient agar (Difco), hexadecane (≥ 99%, Sigma-Aldrich), dioctyl sodium sulfosuccinate (DOSS, ≥ 97%, Sigma-Aldrich), SYTO9 (ThermoFisher), carbonyl cyanide 3chlorophenylhydrazone (CCCP, ≥ 97%, Sigma-Aldrich), sodium chloride (≥ 99%, BDH), magnesium chloride hexahydrate (≥ 99%, Alfa Aesar), magnesium sulfate heptahydrate (≥ 99.5%, Sigma-Aldrich), calcium chloride dihydrate (Sigma-Aldrich), potassium chloride (≥ 99%, BDH), potassium nitrate (≥ 99%, EMD), dipotassium phosphate (≥ 98%, Sigma-Aldrich), ethylene glycol (≥ 99.8%, Sigma-Aldrich), and diiodomethane (≥ 99, Sigma-Aldrich) were used as received.

Dewangan N.K, & Conrad J.C. (2020). Bacterial motility enhances adhesion to oil droplets. Soft matter, 16(35).

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Viral Plaque Assay Protocol

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Vero cells were plated in 12-well plates at a density of 2 × 10⁵ cells/well for 24 h. Viral stocks or infection supernatants were serially diluted to 10⁻⁸ in culture media and overlaid on cells for 1 h. Cells were covered with Eagle’s Minimum Essential Medium (without phenol red, supplemented with 5% fetal bovine essence, non-essential amino acids, 1 mM sodium pyruvate (45000-710, VWR), 2 mM L-glutamine, 20 U/mL penicillin, and 20 µg/mL streptomycin) containing 0.6% agarose. 48 h post-infection, cells were fixed with 10% formaldehyde (F79P-4, FisherSci) for 1 h. Medium was removed, wells were washed with diH₂O, and cells were stained with a 1% crystal violet (FisherSci, C581-25) in a 20% ethanol solution (BP2818-4, FisherSci). For plaque assay of VEEV TrD nsP3 mutants, virus was serially diluted in virus diluent (phosphate-buffered saline, 1% donor calf serum) and overlaid on BHK-J cells seeded at a density of 1 × 10⁶ cells/well in 6-well plates for 24 h. Plates were stained with neutral red and plaques were counted. All viral entities were titered in triplicate unless otherwise specified.

Bakovic A., Bhalla N., Kortchak S., Sun C., Zhou W., Ahmed A., Risner K., Klimstra W.B, & Narayanan A. (2020). Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKKβ Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis. Viruses, 12(9), 1021.

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Splenic Lymphocyte Isolation and Treatment

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The splenocytes from C57BL/6 mice were isolated by placing the spleen atop of a 40 μm cell strainer and gently pressing a pestle against the spleen to separate the splenocytes. The effluent was centrifuged at 300 RFC for 5 mins. Once the supernatant was removed, 3 mL of 1x red blood cell lysis buffer was added for 5 mins at 4°C. The splenocytes were then centrifuged at 300 RFC for 5 mins. The supernatant was discarded and the cells were resuspended in RPMI-1640 Medium with L-glutamine (VWR, Radnor, PA), 10% fetal bovine serum, 1% sodium pyruvate (VWR, Radnor, PA), 1% non-essential amino acids (VWR, Radnor, PA), and 1% penicillin–streptomycin (VWR, Radnor, PA). The splenocytes were plated at 250,000 cells per well and were treated 2 hrs after the cells were seeded. The final concentration in each well of each treatment was as follows: 0.1 mg/mL of paKG nanoparticles, 1 mg/mL of methotrexate and 2.5 μg/mL of concanavalin A (positive control). No treatment was used as a negative control. The treated splenocytes were cultured for 48-72 hrs.

Mangal J.L., Inamdar S., Suresh A.P., Jaggarapu M.M., Esrafili A., Ng N.D, & Acharya A.P. (2022). Short Term, Low Dose Alpha-ketoglutarate Based Polymeric Nanoparticles with Methotrexate Reverse Rheumatoid Arthritis Symptoms in Mice and Modulate T Helper Cell Responses. Biomaterials science, 10(23), 6688-6697.

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Splenocyte-BMDC Mixed Lymphocyte Reaction

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Spleen of 6–8-week-old BALB/c mice were isolated for mixed lymphocyte reaction (MLR) studies. Cells were extracted from the spleen by applying firm pressure from a pestle against a cell strainer. The effluent was centrifuged at 300 × Gs for 5 min. The supernatant was discarded and the splenocytes were then resuspended in 3 mL of 1x RBC lysis buffer for 3 min at 4 °C. The cells were then centrifuged at 300 × Gs for 5 min and the pellet was resuspended in DMEM/F-12 with L-glutamine (VWR, Radnor, PA), 10% fetal bovine serum, 1% sodium pyruvate (VWR, Radnor, PA), 1% non-essential amino acids (VWR, Radnor, PA), and 1% penicillin–streptomycin (VWR, Radnor, PA). After resuspension, the splenocytes were used in MLR and seeded with BMDCs from DBA/1j mice on day 10 of the BMDC isolation protocol[51 (link)–55 (link)], shortly after the treatment of the cells. The splenocyte to BMDC ratio was 20:1 for 48–72 hrs. No treatment was used as a control.

Mangal J.L., Inamdar S., Le T., Shi X., Curtis M., Gu H, & Acharya A.P. (2021). Inhibition of glycolysis in the presence of antigen generates suppressive antigen-specific responses and restrains rheumatoid arthritis in mice. Biomaterials, 277, 121079.

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Cell Culture and Inhibitor Treatments

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LOX and MDA-MB-231 cells were maintained in RPMI (GIBCO), supplemented with 2mM L-glutamine (Invitrogen), 10% FBS (HyClone), penicillin and streptomycin (Invitrogen). A375ma2 (ATCC) melanoma cells were maintained in high glucose DMEM (Sigma), supplemented with 2mM L-glutamine (Invitrogen), 10% FBS (HyClone), 1mM sodium pyruvate (VWR), penicillin and streptomycin (Invitrogen). Cells were grown in humidified incubator at 37°C with 5% CO₂. For inhibitor experiments, cells were grown in the presence of CEP-1347 (1 μM) purchased from Tocris Bioscience, or PD98059 (25 μg/mL) purchased from Cayman Chemical, or JNK inhibitor II (also known as SP600125) (20 μM) purchased from Calbiochem, for 24 hours. 1μM of 17-AAG inhibitor was used for inhibition of Hsp90 activity.

Balinda H.U., Sedgwick A, & D’Souza-Schorey C. (2022). Mechanisms underlying melanoma invasion as a consequence of MLK3 loss. Experimental cell research, 415(1), 113106.

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Generation of Plasmacytoid Dendritic Cells

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In order to obtain pDC we implemented a modified version of the protocol used in Mildner at al.(Mildner et al., 2013a (link)). Briefly, at day 0, Cells were plated at a density of a million cells/ml on 10 cm non-tissue culture treated plastic dishes using 10ml medium per plate. At day 2, 5 and 7 cells were fed with another 3ml medium per dish. pDC medium contains: RPMI (Invitrogen) supplemented with 10% heat inactivated FBS (Invitrogen), β-mercaptoethanol (50uM, Invitrogen), L-glutamine (2mM, VWR), MEM non-essential amino acids (VWR), HEPES (10mM, VWR), sodium pyruvate (1mM, VWR), and Flt3L (200ng/ml, peprotech).

Bornstein C., Winter D., Barnett-Itzhaki Z., David E., Kadri S., Garber M, & Amit I. (2014). A negative feedback loop of transcription factors specifies alternative dendritic cell chromatin states. Molecular cell, 56(6), 749-762.

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