Pyruvate

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Pyruvate is a key metabolite in cellular respiration and energy production. It serves as a critical intermediate in the tricarboxylic acid (TCA) cycle, also known as the Krebs cycle. Pyruvate is a versatile compound that can be converted into various other biomolecules, making it an important component in a wide range of biochemical processes.

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Lab products found in correlation

379 protocols using pyruvate

Rotenone-Induced Neurotoxicity Mitigation

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All chemicals were prepared as concentrated solutions according to the recommendations of the different manufacturers. Compounds were aliquoted in Eppendorf tubes and used once, to avoid repeated freezing/thawing processes. Aliquots were stored at −80 °C for no longer than two months. Care was taken to protect photosensitive molecules from light by wrapping the test tubes in aluminum foil. Drugs were extemporaneously diluted at their respective final concentration in DMEM containing 10% FBS+ N2+ B27 minus Anti-Oxidant (AO). In order to study the impact of Glucose on Rotenone induced toxicity, two distinct DMEM formulations differing only in their glucose concentration were used. HG conditions correspond to DMEM, high glucose, Glutamax Supplement, Pyruvate (Thermofisher, Gibco, Cat ref 10569010) containing 4.5 g.L⁻¹ (25 mM) of glucose. LG condition corresponds to DMEM, low glucose, Glutamax Supplement, Pyruvate (Thermofisher, Gibco Cat ref 10567014) containing 1 g.L⁻¹ (5 mM) of glucose. Ten days after seeding, Rotenone (5 μM, Sigma-Aldrich) with or without mdivi-1 (20 μM, Tocris) or SP 600125 (10 μM) diluted in LG or HG DMEM were applied for 24 or 48 h according to the treatment. After treatment, neurons were fixed with Paraformaldehyde 4% (Sigma-Aldrich) for 10 minutes at room temperature then washed with PBS.

Lassus B., Magifico S., Pignon S., Belenguer P., Miquel M.C, & Peyrin J.M. (2016). Alterations of mitochondrial dynamics allow retrograde propagation of locally initiated axonal insults. Scientific Reports, 6, 32777.

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Cell Culture Conditions for MDCK, 293T, and E.Derm

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Madin-Darby Canine Kidney (MDCK; ATCC CCL-34) and 293T cells (ATCC CRL-3216) were grown at 37°C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) high glucose, GlutaMax, and pyruvate (ThermoFisher Scientific) supplemented with 10% Fetal Bovine Serum (FBS; Gibco Life Technologies). E.Derm cells (ATCC CCL-57) were grown at 37°C and 5% CO₂ in DMEM high glucose, GlutaMax, and pyruvate supplemented with 15% FBS, and 1% nonessential amino acids (NEAA; Gibco Life Technologies).

Amat J.A., Patton V., Chauché C., Goldfarb D., Crispell J., Gu Q., Coburn A.M., Gonzalez G., Mair D., Tong L., Martinez-Sobrido L., Marshall J.F., Marchesi F, & Murcia P.R. (2021). Long-term adaptation following influenza A virus host shifts results in increased within-host viral fitness due to higher replication rates, broader dissemination within the respiratory epithelium and reduced tissue damage. PLoS Pathogens, 17(12), e1010174.

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Culture Conditions for Cell Lines

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MDCK (ATCC CCL-34) and human embryonic kidney (293T; ATCC CRL-11268) cells were grown at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) high glucose, GlutaMax, and pyruvate (ThermoFisher Scientific) supplemented with 10% fetal calf serum (Gibco Life Technologies) and 1% PS (100 units/ml penicillin, 100 μg/ml streptomycin; Gibco Life Technologies). E-derm cells (ATCC CCL-57) were grown at 37°C and 5% CO₂ in DMEM high glucose, GlutaMax, and pyruvate supplemented with 15% fetal calf serum (Gibco Life Technologies), 1% nonessential amino acids (Gibco, Life Technologies), 1% PS (Gibco Life Technologies).

Chauché C., Nogales A., Zhu H., Goldfarb D., Ahmad Shanizza A.I., Gu Q., Parrish C.R., Martínez-Sobrido L., Marshall J.F, & Murcia P.R. (2018). Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1. Journal of Virology, 92(5), e01875-17.

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Modulation of PBMC Cytokine Production

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After approval from the local ethics committee of the Radboud university medical center (CMO 2010/10), EDTA anticoagulated blood was obtained from 6 healthy donors. Isolation of PBMCs was performed by differential centrifugation over Ficoll-Paque PLUS (GE Healthcare Biosciences) in SepMate tubes (STEMCELL technologies). PBMCs were washed thrice with phosphate buffered saline (PBS), counted, and 5 × 10⁵ PBMCs/well were seeded in 96-well round-bottom plates in Roswell Park Memorial Institute (RPMI) 1640 culture medium (Dutch Modification, Invitrogen) supplemented with 50 μg/mL gentamycin (Thermo Fisher Scientific) and 2 mM Glutamax (Invitrogen). Cells were incubated with RPMI (control), or 1, 3, 10, or 20 mM of sodium lactate (provided by the Department of Pharmacy, Radboud University Medical Center, Nijmegen, The Netherlands), pyruvate (Invitrogen), or a combination of sodium lactate and pyruvate for 1 h at 37 °C and 5% CO₂. Subsequently, RPMI or 10 ng/mL E. coli-derived LPS (serotype O55:B5, Sigma Aldrich) was added and PBMCs were incubated for 48 h at 37 °C and 5% CO₂. Concentrations of IL-10, IL-1β, IL-6, and TNFα in the cell culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA, R&D systems).

Zwaag J., ter Horst R., Blaženović I., Stoessel D., Ratter J., Worseck J.M., Schauer N., Stienstra R., Netea M.G., Jahn D., Pickkers P, & Kox M. (2020). Involvement of Lactate and Pyruvate in the Anti-Inflammatory Effects Exerted by Voluntary Activation of the Sympathetic Nervous System. Metabolites, 10(4), 148.

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Placental BeWo Cell Preparation

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Placental BeWo cells were seeded at 26,300 cells/cm³ on glass coverslips and kept in culture for 24 h with DMEM, high glucose, pyruvate (Gibco)/10% FBS/1% Pent/Strep. Cells were starved in DMEM, high glucose, pyruvate (Gibco)/1% FBS/1% Pent/Strep overnight. Then the cells were incubated with acetic acid buffer (0.2 M acetic acid + 0.5 M NaCl) for 5 min to remove potential serum asprosin from the cell surface. Then cells were fixed with 100% methanol for 10 min and stored in PBS until usage.

Hoffmann T., Morcos Y.A., Janoschek R., Turnwald E.M., Gerken A., Müller A., Sengle G., Dötsch J., Appel S, & Hucklenbruch-Rother E. (2022). Correlation of metabolic characteristics with maternal, fetal and placental asprosin in human pregnancy. Endocrine Connections, 11(3), e220069.

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Culturing GalT-GFP-SNAP26, hTERT-RPE1, and WISH Cells

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All cells were grown at 37 °C under 5% CO₂ and routinely tested for the mycoplasma contamination. HeLa cells stably expressing GalT-GFP-SNAP26 (link) were cultured in DMEM high-glucose Glutamax (Gibco, Life Technologies), supplemented with 10% FCS (v/v) (Gibco, Life Technologies), 5 mM pyruvate (v/v) (Gibco, Life Technologies) and 1% penicillin–streptomycin (v/v) (Gibco, Life Technologies). hTERT-RPE1 (human retinal pigmented epithelial) cells (kind gift of P. Benaroch) were cultured in DMEM/F12 Glutamax (Gibco, Life Technologies), supplemented with 10% FCS. WISH cells (kind gift of D. Novick) were grown in MEM GlutaMAX (Gibco, Life Technologies) supplemented with 10% FCS, 5 mM pyruvate and 1% penicillin–streptomycin. WISH cells, which remain one of the most used cells in the IFN field, allowed to confirm and extend the role of the retromer on JAK/STAT signalling in another cell type. All cell lines were routinely tested for mycoplasma contamination.

Chmiest D., Sharma N., Zanin N., Viaris de Lesegno C., Shafaq-Zadah M., Sibut V., Dingli F., Hupé P., Wilmes S., Piehler J., Loew D., Johannes L., Schreiber G, & Lamaze C. (2016). Spatiotemporal control of interferon-induced JAK/STAT signalling and gene transcription by the retromer complex. Nature Communications, 7, 13476.

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Cell Culture Conditions for Multiple Cell Lines

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HeLa cells, Cav1-EGFP stably transfected HeLa cells (Sinha et al., 2011 (link)), and Hs578T cells were grown at 37°C under 5% CO₂ in DMEM GlutaMAX (GIBCO BRL Life Technologies) supplemented with 10% FCS (GIBCO BRL Life Technologies), 5 mM pyruvate (GIBCO BRL Life Technologies), and 1% penicillin-streptomycin (GIBCO BRL Life Technologies). HeLa His-SUMO2 cells were grown as HeLa cells with 1 µg/ml puromycin (InvivoGen). MLECs (Sinha et al., 2011 (link)) were maintained in EGM-2 medium (Lonza) supplemented with 15% FBS (Hyclone; GE Healthcare), 4 mM l-glutamine (GIBCO BRL Life Technologies), 5 mM pyruvate, and 1% penicillin-streptomycin. MDA-MB-436 cells were grown at 37°C without CO₂ in Leibovitz’s L-15 medium (GIBCO BRL Life Technologies) supplemented with 10% FCS (GIBCO BRL Life Technologies) and 1% penicillin-streptomycin (GIBCO BRL Life Technologies).

Torrino S., Shen W.W., Blouin C.M., Mani S.K., Viaris de Lesegno C., Bost P., Grassart A., Köster D., Valades-Cruz C.A., Chambon V., Johannes L., Pierobon P., Soumelis V., Coirault C., Vassilopoulos S, & Lamaze C. (2018). EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription. The Journal of Cell Biology, 217(12), 4092-4105.

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Intestinal Cell Response to Digesta Exposure

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The transwell inserts and 96-well plates were rinsed with glucose-free DMEM supplemented with 10 mM HEPES buffer, 100 IU/mL penicillin, 100 μg/mL streptomycin and non-essential amino acids (1/100 dilution of 100× solution, ThermoFisher), 10 mM pyruvate (ThermoFisher). The final small intestinal digesta from simulated digestions were combined with glucose-free DMEM media in a ratio of 1:3, and the mixture was applied to the cells (1.5 ml to the apical compartment for transwell inserts, 200 µl per well for 96-well plates). Apical fluid in untreated control wells was replaced with fresh glucose-free media. Digesta was also dispensed in a cell-free control well. Transwell cells were incubated with digesta for 4 h. At the end of exposure, TEER in transwells was measured as described above, and supernatants from transwells were collected for LDH analysis. Assessment of ROS production and cell viability (described below) was performed after 4 h exposures, respectively, in 96-well plates.

Guo Z., DeLoid G.M., Cao X., Bitounis D., Sampathkumar K., Woei Ng K., Joachim Loo S.C, & Philip D. (2021). Effects of ingested nanocellulose and nanochitosan materials on carbohydrate digestion and absorption in an in vitro small intestinal epithelium model. Environmental science. Nano, 8(2), 2554-2568.

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Spermatogonia Isolation and Co-culture

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Testes tissue samples (N = 5) were digested and cultured as described by Pieri and collaborators [22 (link)]. First, germ cells and somatic cells were cultured in Dulbecco’s modified Eagle medium/F12 (Cat#BR-30004-05, LGC, Cotia, Brazil) supplemented with 10% fetal bovine serum, 2 mM l-glutamine (Cat#12657029, Cat#25030-081, respectively; Gibco/Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin/streptomycin, 50 μL of pyruvate (Cat#15140-122, Cat#11360-070, respectively; Thermo Fisher Scientific, Waltham, MA, USA), and 100 U/L amphotericin B (Cat#A2942; Sigma-Aldrich Corp., St. Louis, MO, USA). Second, spermatogonia were purified by using Percoll® (Cat#P1644, Sigma-Aldrich Corp., St. Louis, MO, USA), and cells were collected from the 27–35% fractions. Third, the cells were resuspended in the culture medium, plated and incubated at 38 °C under 5% CO₂ [22 (link)].
Finally, the cSSCs were co-cultured with Sertoli cells and treated with 10 IU L⁻¹ FSH (Cat#4021, Sigma-Aldrich Corp., St. Louis, MO, USA) [23 (link)]. These cells were trypsinized and collected after 72 and 120 h of in vitro treatment and were separated into two groups: one supplemented with FSH and another without FSH (control).

Pieri N.C., Mançanares A.C., de Souza A.F., Fernandes H., Diaza A.M., Bressan F.F., Roballo K.C., Casals J.B., Binelli M., Ambrósio C.E, & dos Santos Martins D. (2019). Xenotransplantation of canine spermatogonial stem cells (cSSCs) regulated by FSH promotes spermatogenesis in infertile mice. Stem Cell Research & Therapy, 10, 135.

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Effects of Glucose and Serum on Cell Growth

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In order to establish whether there is a significant effect of glucose concentration and/or source of sera on cell growth, cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco™, Thermo Fisher, Loughborough, UK) low glucose (1 g/L) or with DMEM high glucose (4.5 g/L; Gibco™, Thermo Fisher UK), both supplemented with GlutaMAX (3.97 mM; Thermo Fisher, Loughborough, UK) and pyruvate (1 mM, ThermoFisher, UK). Media was completed with 10% foetal bovine serum (FBS) from one of two different sources/suppliers, designated A and B, both serum sources had a comparable source of origin (USA; please see Appendix B for supplier information).
In total, each cell line was cultured in four different growth medium compositions across a period of 7 days. Each of the four variants of growth media were designated as follows:
DMEM low glucose (1 g/L) supplemented with 10% FBS from source A (LG-A).
DMEM high glucose (4.5 g/L) supplemented with 10% FBS from source A (HG-A).
DMEM low glucose (1 g/L) supplemented with 10% FBS from source B (LG-B).
DMEM high glucose (4.5 g/L) supplemented with 10% FBS from source B (HG-B).

Brown M.J., Bahsoun S., Morris M.A, & Akam E.C. (2019). Determining Conditions for Successful Culture of Multi-Cellular 3D Tumour Spheroids to Investigate the Effect of Mesenchymal Stem Cells on Breast Cancer Cell Invasiveness. Bioengineering, 6(4), 101.

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