Sodium pyruvate

Manufactured by Eurobio Scientific

Sourced in France

Sodium pyruvate is a chemical compound used in cell culture media. It serves as an energy source and metabolic intermediate for cells in vitro.

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14 protocols using sodium pyruvate

Cell Culture and Cytotoxicity Assay

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HEK293T and Jurkat cell lines (ATCC) were maintained either with DMEM or RPMI1640 medium containing 10% of FBS, penicillin/streptomycin antibiotics, and l-glutamine.
For the measurement of target lysis, K562 cells were first cultured overnight in RPMI1640 without phenol red (Gibco) enriched with 10% FBS, non-essential amino acids (NEAA, Gibco), l-glutamine (Life Technologies) and Sodium Pyruvate (Eurobio). Prior killing assay, K562 cells were diluted at 1 × 10⁶/mL in a culture medium supplemented with 2.5 mM Probenecid (Invitrogen) and stained with calcein-AM (Invitrogen) for 30 min. Then, the excess calcein was washed out.
For in vitro cell treatment, PBMCs were resuspended in RPMI 1640 supplemented with 10% human serum (EFS, Etablissement Français du Sang), 1% Penicillin–Streptomycin (Dutscher), l-glutamine (Life Technologies), Sodium Pyruvate (EuroBio) and HEPES (EuroBio).

Boy M., Bisio V., Zhao L.P., Guidez F., Schell B., Lereclus E., Henry G., Villemonteix J., Rodrigues-Lima F., Gagne K., Retiere C., Larcher L., Kim R., Clappier E., Sebert M., Mekinian A., Fain O., Caignard A., Espeli M., Balabanian K., Toubert A., Fenaux P., Ades L, & Dulphy N. (2023). Myelodysplastic Syndrome associated TET2 mutations affect NK cell function and genome methylation. Nature Communications, 14, 588.

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Isolation and Propagation of Metastatic Tumor Cells

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Metastatic cells were obtained as reported by Pulaski,²⁷ with minor changes. BALB/c mice were inoculated with 1 × 10⁴cells into the fourth mammary fat pad, allowing primary tumor development.
Twenty-five days later, the mice were humanely euthanized by CO₂inhalation. First, the primary tumor, lungs, and liver were resected.
Subsequently, the organs and tumor were transferred to RPMI-1640 media (Eurobio)
supplemented with 2% penicillin/streptomycin (Eurobio). Next, the tissues were
sliced into small pieces and digested with 2.5 mL of type-IV collagenase (Gibco,
Life Technologies, New York, NY) in RPMI-1640 media supplemented with 10 mM
HEPES, 2.5% fetal bovine serum (FBS), 2% penicillin/streptomycin, 1 mM sodium
pyruvate, and 2 mM L-glutamine (Eurobio); lung and liver digestion was carried
out at 4°C for 90 minutes. Subsequently, the tissues were filtered by a 70 µm
cell strainer and centrifuged for 5 minutes at 300g, and after
the lysis of red blood cells, the cells were cultured in the presence of
6-thioguanine (Sigma-Aldrich, St. Louis, MO) to select metastatic tumor cells.
Once colonies were observed, attached cells were recovered with 0.25%
trypsin/0.02% EDTA (Eurobio), and then the cells were washed with supplemented
RPMI media and transferred to T-25 culture flasks. Metastatic cells were
obtained from at least 3 different mice during 3 serial in vivo passages.

Lasso P., Llano Murcia M., Sandoval T.A., Urueña C., Barreto A, & Fiorentino S. (2019). Breast Tumor Cells Highly Resistant to Drugs Are Controlled Only by the Immune Response Induced in an Immunocompetent Mouse Model. Integrative Cancer Therapies, 18, 1534735419848047.

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Cell Culture in RPMI-1640 Media

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B16-F10, 4T1, Mel-Rel, TS/A, MCF-7, MDA-MB-468, LAU 145, and Me290 cell lines were cultured in RPMI-1640 (Eurobio, Toulouse, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37°C and 5% CO₂. Cells were grown until 75% confluency and passaged using trypsin/1× EDTA (Eurobio), washed with PBS, and resuspended in supplemented RPMI-1640.

Lasso P., Gomez-Cadena A., Urueña C., Donda A., Martinez-Usatorre A., Romero P., Barreto A, & Fiorentino S. (2020). An Immunomodulatory Gallotanin-Rich Fraction From Caesalpinia spinosa Enhances the Therapeutic Effect of Anti-PD-L1 in Melanoma. Frontiers in Immunology, 11, 584959.

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Culturing and Analyzing DA-3/ER-GM Cells

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The DA-3/ER-GM cells were kindly provided by Prof. Jose Arteaga (Departament of Basic Science, Medical School, Universidad Industrial de Santander, Bucaramanga, Colombia). DA-3/ER-GM cells were cultured in RPMI-1640 medium (Eurobio, Toulouse, France) with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicilin, 100 μg/mL streptomycin, 0.01 M HEPES buffer, 1 mM sodium pyruvate (Eurobio), and 50 pg/mL gentamycin (Gibco, Grand Island, NY, USA) and cultivated at 37 °C in 5% CO₂. Evaluation of cell morphology was performed with 2 × 10⁵ cells in Cytospin preparations and stained, using the Wright stain, prior to photographing using 100× magnification.

Murillo N., Lasso P., Urueña C., Pardo-Rodriguez D., Ballesteros-Ramírez R., Betancourt G., Rojas L., Cala M.P, & Fiorentino S. (2023). Petiveria alliacea Reduces Tumor Burden and Metastasis and Regulates the Peripheral Immune Response in a Murine Myeloid Leukemia Model. International Journal of Molecular Sciences, 24(16), 12972.

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Culture and Maintenance of Leukemic Cell Lines

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The human leukemic cell line K562 (from a patient with chronic myeloid leukemia (CML)) was obtained from American type culture collection (Manassas, VA, USA). The human leukemic cells lines U937 (from a patient with monocytic differentiation-inducible histiocytic lymphoma) and Jurkat (from the peripheral blood of a 14-year-old boy with T-cell acute lymphoid leukemia) were provided by Dr. Hélène Moins‐Teisserenc (Laboratoire d'Hématologie Biologique, AP‐HP, Hôpital Saint Louis, 75010 Paris, France). Cells were grown in RPMI-1640 (Eurobio, Toulouse, France) supplemented with heat-inactivated fetal bovine serum (10%; Eurobio), 2 mM L-glutamine, 100 U/mL penicillin, 100 ug/mL streptomycin, 0.01 M Buffer Hepes, and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37°C and 5% CO₂. Cell lines were proven mycoplasma-free using a MycoProbe Mycoplasma Detection Kit (R&D Systems) and maintained with ciprofloxacin (0.5 μg/mL).

Ballesteros-Ramírez R., Aldana E., Herrera M.V., Urueña C., Rojas L.Y., Echeverri L.F., Costa G.M., Quijano S, & Fiorentino S. (2020). Preferential Activity of Petiveria alliacea Extract on Primary Myeloid Leukemic Blast. Evidence-based Complementary and Alternative Medicine : eCAM, 2020, 4736206.

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Characterizing Murine Tumor Cell Lines

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The murine mammary tumor cell line 4T1 (a gift from Alexzander Asea of Texas A&M Health Science Center College of Medicine, Temple, TX, USA), murine fibroblasts 3T3 cell line (ATCC® CRL-1658™), and HPdLF Human Periodontal Ligament Fibroblasts (Lonza CC-7049) were cultured in RPMI-1640 (Eurobio, Les Ulis Cedex B, France) supplemented with heat-inactivated fetal calf serum (10%) (Eurobio), 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/ml streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37 °C and 5% CO₂. Cells were grown until 75% confluence and passaged using trypsin/EDTA 1X (Eurobio), washed with phosphate-buffered saline 1X (PBS), and resuspended in supplemented RPMI-1640. Cells were treated with murine TGFβ (Cell Signaling Technology, Beverly, MA, USA) and RSV (Sigma-Aldrich, St. Louis, MO-USA). P2Et half maximal inhibitory concentration (IC₅₀) for 3T3 (142.7 μg/ml) and 4T1 (34.1 μg/ml) was obtained by MTT assay (Supplementary Fig. 2).

Jiménez M.C., Prieto K., Lasso P., Gutiérrez M., Rodriguez-Pardo V., Fiorentino S, & Barreto A. (2023). Plant extract from Caesalpinia spinosa inhibits cancer-associated fibroblast-like cells generation and function in a tumor microenvironment model. Heliyon, 9(3), e14148.

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Culturing Human Dermal Microvascular and Brain Endothelial Cells

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The human dermal microvascular endothelial HMEC‐1 cell line, provided by A. Kesikli (University of Regensburg, Germany) was cultured in complete MCDB 131 medium, composed of MCDB 131 medium (Thermofisher Scientific) supplemented with 12.5% fetal bovine serum (FBS), hydrocortisone 1 μg/ml (Sigma‐Aldrich), epidermal growth factor (EGF) 10 ng/ml (BD Biosciences), 6 mM Glutamine and used between passages 12 and 20.¹⁹, ²⁰ The hCMEC/D3 cell line was cultured in complete EndoGRO Basal Medium (Merck), composed of EndoGRO Basal Medium supplemented with 0.2% EndoGRO LS Supplement, recombinant EGF 5 ng/ml, hydrocortisone hemisuccinate 1 μg/ml, heparin sulfate 0.75 U/ml, ascorbic acid 50 μg/ml, l‐Glutamine 10 mM, 5% FBS and used between passage 30 and 40.²⁶ Coculture experiments were carried out with ECs and peripheral blood mononuclear cells (PBMCs), as reported elsewhere.¹⁹, ²⁰ Briefly, PBMCs were isolated from healthy donor blood samples (obtained in accordance with institutional regulations from the Etablissement Français du Sang) by Ficoll density gradient separation (Eurobio). PBMCs were maintained in complete RPMI 1640 medium (Thermofisher Scientific), composed of RPMI 1640 with 10% human AB serum and a final concentration of HEPES 10 mM, sodium pyruvate 1× (Eurobio), glutamine 2 mM.

Pons S., Arrii E., Arnaud M., Loiselle M., Ferry J., Nouacer M., Lion J., Cohen S., Mooney N, & Zafrani L. (2021). Immunomodulation of endothelial cells induced by macrolide therapy in a model of septic stimulation. Immunity, Inflammation and Disease, 9(4), 1656-1669.

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Analytical Reagents for Cell Culture

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Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin sulphate and sodium pyruvate were obtained from Eurobio (Les Ulis, France). Fetal calf serum, isoflurane and nonessential amino acids were purchased from Seromed Biochrom (Berlin, Germany), AErrane (Baxter S.A., Belgium) and Cambrex (Walkersville, MD, USA), respectively. Other chemicals, such as trichloroacetic acid (TCA), quercetin, ferric chloride, potassium ferricyanide (K₃Fe(CN)₆), gallic acid, etc., were of analytical grade and purchased from Sigma-Aldrich.

Badraoui R., Rebai T., Elkahoui S., Alreshidi M., N. Veettil V., Noumi E., A. Al-Motair K., Aouadi K., Kadri A., De Feo V, & Snoussi M. (2020). Allium subhirsutum L. as a Potential Source of Antioxidant and Anticancer Bioactive Molecules: HR-LCMS Phytochemical Profiling, In Vitro and In Vivo Pharmacological Study. Antioxidants, 9(10), 1003.

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Murine Melanoma and Breast Cancer Cell Lines

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The murine melanoma B16F10 cell line was kindly provided by PR (Ludwig Center for Cancer Research, Department of Oncology—Faculty of Biology and Medicine University of Lausanne, Switzerland). The B16 mouse model was created in the 1970s from a melanoma that developed spontaneously in the ear of a female C57BL/6 mouse and was then passaged in vivo to create the B16F10 line (21 (link)). The murine breast cancer 4T1 cell line was grown in BALB/c mice and developed into a highly tumorigenic and invasive tumor that can spontaneously metastasize from a primary tumor in the mammary gland to multiple distant sites (22 (link)). The 4T1 mammary tumor cells were kindly provided by Dr Alexzander Asea (Texas A&M Health Science Center College of Medicine, Temple, TX). Cells were cultured in RPMI-1640 (Eurobio, Toulouse, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Eurobio), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M HEPES buffer and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37°C and 5% CO₂. Cells were grown until 75% confluency and passaged using trypsin/1X EDTA (Eurobio), washed with PBS and resuspended in supplemented RPMI-1640.

Lasso P., Gomez-Cadena A., Urueña C., Donda A., Martinez-Usatorre A., Barreto A., Romero P, & Fiorentino S. (2018). Prophylactic vs. Therapeutic Treatment With P2Et Polyphenol-Rich Extract Has Opposite Effects on Tumor Growth. Frontiers in Oncology, 8, 356.

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Cell Culture Conditions for 4T1 and B16-F10

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4T1 and B16-F10 cells were cultured in RPMI-1640 medium (Eurobio, Toulouse, France) with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate (Eurobio) and cultivated in a humidified incubator at 37 °C in 5% CO₂.

Lasso P., Rojas L., Arévalo C., Urueña C., Murillo N., Barreto A., Costa G.M, & Fiorentino S. (2022). Tillandsia usneoides Extract Decreases the Primary Tumor in a Murine Breast Cancer Model but Not in Melanoma. Cancers, 14(21), 5383.

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