L glucose

Manufactured by Thermo Fisher Scientific

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L-glucose is a stereoisomer of the naturally occurring D-glucose. It serves as a valuable tool in biochemical research and scientific applications, providing a means to study glucose metabolism and related processes. The core function of L-glucose is to act as a reference compound, allowing researchers to differentiate and analyze the behavior of glucose and its derivatives in various experimental settings.

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DMEM 1 g/L glucose DMEM) Glutamax 4.5 g/l glucose 4.5 g/L D-glucose D-Glucose and L-Glutamine Dulbecco’s modified Eagle’s high glucose w/Glutamax (4.5 g/L) cell culture medium Dulbecco's modified Eagle medium (DMEM with glucose 4.5 g L−1), Dulbecco’s modified Eagle’s medium low glucose 1 g/L (DMEM, DMEM low glucose (1 g/L) DMEM (4.5 g/L d-glucose; DMEM with high glucose and L-glutamine

7 protocols using l glucose

Osteosarcoma Cell Line Cultivation

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The human osteosarcoma cell line Saos-2 (TP53-null) was maintained as a monolayer in Dulbecco's modified Eagle's medium with l-Glucose (ThermoFisher Scientific) supplemented with 10% fetal bovine serum, MEM Non-Essential Amino Acids solution (ThermoFisher Scientific), 2.5 mmol/l-Glutamine and penicillin-streptomycin at 37°C under 5% CO₂. Cells were seeded at a density of 1 × 10⁶ in 10-cm² plates and used for luciferase promoter–reporter, western blotting, and colony reduction assays.

Quinn E.A., Maciaszek J.L., Pinto E.M., Phillips A.H., Berdy D., Khandwala M., Upadhyaya S.A., Zambetti G.P., Kriwacki R.W., Ellison D.W., Nichols K.E, & Kesserwan C. (2019). From uncertainty to pathogenicity: clinical and functional interrogation of a rare TP53 in-frame deletion. Cold Spring Harbor Molecular Case Studies, 5(4), a003921.

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Cell Culture and Transfection Protocol

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COS-7 [34] (link)–[36] (link) and HEK cells [35] (link), [37] (link) were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) containing L-glutamine, penicillin, streptomycin, and 10% (v/v) fetal bovine serum. Cells were transfected using Lipofectamine 2000 reagent (Invitrogen) as described previously[34] (link)–[36] (link), [38] (link), [39] (link). The HA-Nox5 HEK293 cell line was generated by using Flp Recombinase-Mediated Integration (Invitrogen)[36] (link), [37] (link). Human lung microvascular endothelial cells (HLMVEC) were purchased from Lonza, and were grown in Endothelial Growth Medium-2-Microvessel (EGM-2MV) consisting of defined growth factors and supplemented with additional FBS up to 5% final concentration (Lonza). Cells were grown at 37°C in 5% CO₂ incubator and used from passage 2–6.
Ro 32-0432 (Bisindolylmaleimide XI hydrochloride) and Gö 6976 were obtained from Sigma-Aldrich (St.Louis, MO). L-glucose and D-glucose were purchased from Thermo Fisher Scientific (Waltham, MA).

Chen F., Yu Y., Haigh S., Johnson J., Lucas R., Stepp D.W, & Fulton D.J. (2014). Regulation of NADPH Oxidase 5 by Protein Kinase C Isoforms. PLoS ONE, 9(2), e88405.

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INT-407 Cell Culture and Infection

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Cell culture studies using INT-407 cells (human embryonic intestine, ATCC CCL 6; http://www.atcc.org/products/all/CCL-6.aspx) were approved by The Institutional Biosafety Committee, The Ohio State University, under the protocol number 2007R0009AR4. For cell culture, INT-407 cells were grown in Dulbecco's minimal essential medium (DMEM) supplemented with 4 mM L-glutamine, 4.5 g/L L-glucose, and 10% fetal bovine serum (Thermo Scientific, South Logan, UT, USA) at 37 °C in a 5% CO₂ humidified incubator. Cells on monolayers were treated with trypsin (1% trypsin, Gibco, Grand Island, NY, USA), suspended in 24 well tissue culture plates and incubated until confluent monolayers were obtained. To assess the number of INT-407 cells prior to infection, two extra wells were seeded, and the average cell number per well was determined by staining the cells with trypan blue and counting the cells with a hemocytometer under a microscope. For infection, ∼2 × 10⁶ cells per/well were used. Antibiotics were not used for culturing the INT-407 cells in this study.

Pina-Mimbela R., Madrid J.A., Kumar A., Torrelles J.B, & Rajashekara G. (2015). Polyphosphate kinases modulate Campylobacter jejuni outer membrane constituents and alter its capacity to invade and survive in intestinal epithelial cells in vitro. Emerging Microbes & Infections, 4(12), e77-.

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Oxygen-Glucose Deprivation Neuronal Injury Model

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Cells in control group were washed with phosphate buffered saline (PBS) and incubated in neurobasal medium in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. The exposure of cells to OGD was performed as we described before (Wu, et al., 2010 (link)). Briefly, neurobasal-A medium that did not contain L-glucose (GIBCO) was bubbled with 100% N₂ for 30 min. Cells were washed with PBS once and 2 ml/well of the neurobasal-A medium was added to the cells. These plates were immediately placed in an air-tight chamber (Billups-Rothenberg, Inc., Del Mar, CA) gassed with 100% N₂ for 10 min. The oxygen content in the outlet of the chamber was monitored with a DatexTM infrared analyzer (Capnomac, Helsinki, Finland) and reached 2% at 3 – 5 min after the onset of gassing. After closure of the inlet and outlet of the chamber, the chamber was kept at 37°C for 1 h. After confirming that the oxygen content in the chamber was still lower than 2% at the end of the OGD period, the chamber was opened and glucose, B-27 supplement and L-glutamine (500 μM) was added to make the final glucose concentration in the buffer at 4.5 g/l. The plates were kept for 6 h (Western blotting) or 20 h (Western blotting and LDH release assay) in a humidified atmosphere of 95% air and 5% CO₂ at 37°C.

Cheng A., Lu Y., Huang Q, & Zuo Z. (2019). Attenuating oxygen-glucose deprivation-caused autophagosome accumulation may be involved in sevoflurane postconditioning-induced protection in human neuron-like cells. European journal of pharmacology, 849, 84-95.

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Glucose-6-Phosphate Quantification in Cells

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HOS and U2OS cells were incubated with L-arginine (cat. no. A0013; Beijing Solarbio Science & Technology Co., Ltd.) and glucose-free F-12K (cat. no. 21127022, Gibco; Thermo Fisher Scientific, Inc.) medium for 30 min at 37°C and then incubated with 7 mM L-glucose (cat. no. 20829; Cayman Chemical Company), 1 mM L-arginine, or 1 mM D-arginine (cat. no. A769515; Toronto Research Chemicals) for another 30 min at 37°C. Cell lysates were then collected and glucose 6-phosphate content was determined using a Glucose-6-Phosphate Fluorometric Assay kit (cat. no. 700750-96 wells; Cayman Chemical Company). Finally, the concentration of glucose 6-phosphate in cells was determined according to the methods reported in the literature (27 (link)).

Pu F., Liu J., Jing D., Chen F., Huang X., Shi D., Wu W., Lin H., Zhao L., Zhang Z., Lv X., Wang B., Zhang Z, & Shao Z. (2022). LncCCAT1 interaction protein PKM2 upregulates SREBP2 phosphorylation to promote osteosarcoma tumorigenesis by enhancing the Warburg effect and lipogenesis. International Journal of Oncology, 60(4), 44.

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Mitochondrial Energy Production in HepG2-ERα Cells

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HepG2-ERα cells were seeded at a density of 5 × 10⁴ in William’s medium (Gibco, Waltham, MA, USA) with phenol red. The next day, HepG2-ERα cells were treated in triplicate using Veh, 1 μM PaPE-1, 100 nM OA with/without 1 μM PaPE-1 in corresponding treatment media without phenol red in each well of the XFp Cell Culture miniplates, respectively (Seahorse Bioscience Inc., Billerica, MA, USA) for 24 h. Concentrations of ligands are based on our previously published study (12). The cartridges were hydrated with the calibration solution and kept in a non-CO₂ incubator at 37 °C overnight. In parallel, a duplicate of each plate was used for cell counting to monitor cell number changes after 24 h of treatments, and Seahorse data was normalized to total cell number. On the assay day, cells were washed with XF Base Media without phenol red supplemented with 10 mM L-glucose, 2 mM L-glutamine (Gibco, Waltham, MA, USA), and 1 mM sodium pyruvate (Gibco, Waltham, MA, USA) and incubated in the same media for 45–60 min before assays were run with Seahorse XFp Analyzer (Seahorse Bioscience Inc., North Billerica, MA, USA). Mitochondrial energy production was measured using the Seahorse XFp Cell Mito Stress Test Kit (Seahorse Bioscience Inc., North Billerica, MA, USA). Experiments were performed in triplicate and repeated at least three times.

Zuo Q., Chen K.L., Arredondo Eve A., Liu Y.J., Kim S.H., Katzenellenbogen B.S., Katzenellenbogen J.A, & Madak-Erdogan Z. (2021). Pathway Preferential Estrogens Prevent Hepatosteatosis Due to Ovariectomy and High-Fat Diets. Nutrients, 13(10), 3334.

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Metabolic Profiling of ESR1-Mutant Breast Cells

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Seahorse XFp plates were coated with Native Coat ECMs for bone, lung, or liver (Xylyx), and were incubated at 37°C in a humidified environment with 5% CO₂ for at least for 1 hour. MCF7-ESR1^Y537S cells were seeded at a density of 3×10⁴ in corresponding treatment media without phenol red in each well of the XFp Cell Culture miniplates (Seahorse Bioscience Inc., Billerica, MA, USA). The next day, the cartridges were hydrated with the calibration solution and were kept overnight in a non-CO₂ incubator at 37°C. To ensure ECAR and OCR values were normalized to the cell number, we monitored the cell number changes after 24 h of treatments. On the assay day, XF Base Media without phenol red (Seahorse Bioscience Inc., Santa Clara, CA) supplemented with 10 mM L-glucose, 2 mM L-glutamine (Gibco), and 1 mM sodium pyruvate (Gibco, Waltham, MA) was used to wash the cells. ECAR (mpH/min) and OCR (pmol/min) values were obtained using Seahorse XFp Cell Energy Phenotype Test Kit (Seahorse Bioscience Inc.). Experiments were performed in triplicate and repeated at least three times.

Zuo Q., Mogol A.N., Liu Y.J., Casiano A.S., Chien C., Drnevich J., Imir O.B., Kulkoyluoglu-Cotul E., Park N.H., Shapiro D.J., Park B.H., Ziegler Y., Katzenellenbogen B.S., Aranda E., O’Neill J.D., Raghavendra A.S., Tripathy D, & Erdogan Z.M. (2022). Targeting metabolic adaptations in the breast cancer–liver metastatic niche using dietary approaches to improve endocrine therapy efficacy. Molecular cancer research : MCR, 20(6), 923-937.

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