Glucose free dmem

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Glucose-free Dulbecco's Modified Eagle Medium (Glucose-free DMEM) is a cell culture medium formulation that does not contain glucose. This medium is designed to support the growth and maintenance of various cell types in an in vitro environment while allowing for the study of glucose metabolism and other cellular processes in the absence of glucose.

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135 protocols using glucose free dmem

Glucose Metabolism in C2C12 Myotubes

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Glucose consumption and lactate production assay in differentiated C2C12 myotubes were performed as previously described (Meng et al., 2013 (link)). Briefly, Cells grown in 6-well plates were glucose deprived in glucose free DMEM (Gibco, 11966) containing 0.1% BSA and 1 mM glucose for 12 h. Then, cells were treated with fresh glucose free DMEM plus 0.1% BSA and certain concentrations of glucose for 24 h. At the end of the treatment, culture medium was replaced with glucose free DMEM plus 0.1% BSA and 5 mM glucose. At 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after incubation, 50 ul of culture medium was collected from each well and frozen in −80°C for fu ture lactate and glucose measurements using Lactic acid assay kit (K-LATE, Megazyme International Ireland Ltd.) and Glucose assay kit (GAGO-20, Sigma), respectively. For ex vivo culture, intact plantaris, soleus, and EDL muscles were dissected from male flox/flox and MKO mice, and subjected to different concentrations of gluocse treatment for 4 h. At the end of the treatment, culture medium was replaced with glucose free DMEM plus 0.1% BSA and 5 mM glucose. At 1 h, 2 h, 3 h after incubation, 50 ul of culture medium was collected for future lactate and glucose measurements.

Meng Z.X., Gong J., Chen Z., Sun J., Xiao Y., Wang L., Li Y., Liu J., Shawn Xu X.Z, & Lin J.D. (2017). Glucose sensing by skeletal myocytes couples nutrient signaling to systemic homeostasis. Molecular cell, 66(3), 332-344.e4.

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Modeling Oxygen-Glucose Deprivation in Cortical Neurons

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Seven days after plating, the primary cortical neurons were washed twice with glucose‐free DMEM (Gibco) and the medium was replaced with glucose‐free DMEM containing L‐glutamine (Gibco) that had been deoxygenated with an anaerobic gas mixture (95% N₂ – 5% CO₂) for 30 min before use. The cells were then placed in a hypoxic chamber, flushed with the anaerobic gas mixture (95% N₂ – 5% CO₂) and incubated at 37°C for 3 hr to establish cell model of oxygen–glucose deprivation (OGD). After OGD, the cells were incubated under normal culture conditions for 0 hr or 24 hr. Neurons in the normal group were treated without OGD exposure.

Ma Z., Wang F., Xue L., Niu Y., Hu Y., Su Z., Huang J., Niu R., Wang T., Ba Y., Xiong L, & Bai X. (2020). bFGF promotes neurological recovery from neonatal hypoxic–ischemic encephalopathy by IL‐1β signaling pathway‐mediated axon regeneration. Brain and Behavior, 10(8), e01696.

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Cell Metabolism Assessment via Resazurin Reduction

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WT, NSUN2 KO and NSUN3 KO HEK293T cells were seeded on 96-well plates (1.0 × 10⁵ cells/well) and cultured in glucose-free DMEM (Gibco) containing 10% FBS, 1% PS, 1 mM sodium pyruvate, and 4% glucose or in glucose-free DMEM (Gibco) containing 10% FBS, 1% PS, 1 mM sodium pyruvate and 4% galactose. On each day, 1/10 volume of 1 mM resazurin solution in PBS was added to each well and incubated for 3 h. Absorbance was measured at 570 and 600 nm on a microplate reader (SpectraMax Paradigm, Molecular Devices). The reduction rate of resazurin was calculated using the following equation: where

E_ox1 = molar extinction coefficient (E) of oxidized (ox) resazurin at 570 nm = 80 586,

E_ox2 = E of oxidized resazurin at 600 nm = 117 216,

E_red1 = E of reduced (red) resazurin at 570 nm = 155 677,

E_red2 = E of reduced resazurin at 600 nm = 14 652,

A1 = absorbance of measured well at 570 nm,

A2 = absorbance of measured well at 600 nm,

C1 = absorbance of blank well (medium and resazurin only) at 570 nm and

C2 = absorbance of blank well at 600 nm.

Shinoda S., Kitagawa S., Nakagawa S., Wei F.Y., Tomizawa K., Araki K., Araki M., Suzuki T, & Suzuki T. (2019). Mammalian NSUN2 introduces 5-methylcytidines into mitochondrial tRNAs. Nucleic Acids Research, 47(16), 8734-8745.

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Cardiomyocyte Ischemia and miR-24 Transfection

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Cardiomyocytes in the logarithmic phase were randomly divided into three groups: i) Cells in the negative control (NC) group were cultured in normal DMEM; ii) cells in the model group were cultured in glucose-free DMEM (Thermo Fisher Scientific, Inc.) with a mixture of 5% CO₂ and 95% N₂ for 10 h at 37˚C under ischemia, followed by addition of high-glucose (4,500 mg/l) DMEM for 2 h of routine culture and iii) cells in the miRNA group were incubated in glucose-free DMEM with a mixture of 5% CO₂ and 95% N₂ gas for 10 h at 37˚C under ischemia, followed by addition of normal DMEM for 2 h of routine culture after transfection of miR-24(1 (link)). Briefly, H9C2 cells were seeded in a 6-well plate at a density of 1x10⁵ cells/well. Once cell fusion reached 70%, Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect cells in the NC and miR-24 (Sangon Biotech Co., Ltd.) groups according to the kit instructions. miRNA NC sequence, 5'-CCTTGGATGGCCTAGGAGATAG-3'; and miR-24 mimic sequence, 5'-TGGCTCAGTTCAGCAGGAACAG-3'. The cells were incubated in an incubator at 37_˚_C and 5% CO₂ for 48 h before subsequent experiments.

Li C., Fang M., Lin Z., Wang W, & Li X. (2021). MicroRNA-24 protects against myocardial ischemia-reperfusion injury via the NF-κB/TNF-α pathway. Experimental and Therapeutic Medicine, 22(5), 1288.

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In vitro Ischemia-Reperfusion Model

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Ischaemia was mimicked in vitro as follows: first, cells were washed with PBS (Solarbio, Beijing, China). Next, the cells were cultured in glucose‐free DMEM (GIBCO, New York, USA) without FBS in a hypoxia incubator (95% N₂, 5% CO₂) at 37°C for 10 hours. After OGD exposure, the medium was replaced with absolute medium containing 10% FBS and cultured under normoxic conditions (95% air, 5% CO₂, 21% FiO₂) to perform OGD/R model. Cells were incubated at 37°C in normal‐glucose DMEM under normoxic conditions as a control. Importantly, the hypoxic chamber was previously sealed and placed in the 37°C thermostat container (SPX‐150C, Boxun, Shanghai, China), where it was flushed in advance with a gas mixture of 95% N₂, 5% CO₂ for 30 min at the rate of 2 L/min. After flushing, the concentration of O₂ was managed with a gas monitor (Smart Sensor, Hong Kong, China). The concentration of O₂ was maintained at less than 1%.

Wang C., Qu Y., Suo R, & Zhu Y. (2019). Long non‐coding RNA MALAT1 regulates angiogenesis following oxygen‐glucose deprivation/reoxygenation. Journal of Cellular and Molecular Medicine, 23(4), 2970-2983.

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Oxygen Glucose Deprivation in Hippocampal Neurons

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Oxygen glucose deprivation was performed as previously described with slight modification (19 (link)). Briefly, the primary hippocampal neurons were washed with glucose-free DMEM (Gibco, USA). Then, after the addition of glucose-free DMEM, they were placed in an anaerobic chamber containing 5% CO₂ and 95% N₂ at 37°C. We sealed cultures inside a modular chamber (Billups-Rothenberg) flushed for 10 min with the same premixed gas and placed inside an incubator for 3 h. OGD was terminated by replacing the exposure medium with neuronal culture medium added with Tat-HA-NR2B9c 250 nM, and returning the cells to a normoxic incubator to allow reperfusion for another 24 h. The cells cultured in the plain medium with ambient oxygen served as control (no exposure to OGD).

Zhou H.H., Zhang L., Zhang H.X., Zhang J.P, & Ge W.H. (2017). Chimeric Peptide Tat-HA-NR2B9c Improves Regenerative Repair after Transient Global Ischemia. Frontiers in Neurology, 8, 509.

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Neuroprotective Effects of Autophagy in Mice

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Pregnant C57BL/6J mice (17–18 days of gestation) and adult mice (6–8 weeks old, 25–30 g) were purchased from the Experimental Animal Center of Peking Union Medical College. Experiments were approved by the Institutional Animal Care and Use Committee of Tianjin Huanhu Hospital. l-Glutamine, 3-methyladenine (3-MA), poly-d-lysine, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit were purchased from Sigma Company (St. Louis, MO, USA). Rabbit anti-mouse LC3, Akt, and phospho-Akt antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Enhanced chemiluminescence (ECL) reagents were purchased from Millipore Company (USA). Fetal bovine serum (FBS), neurobasal medium, B-27 serum-free supplement, Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium, trypsin, rapamycin, and wortmannin were purchased from Life Technologies (Carlsbad, CA, USA). The Akt inhibitor GDC-0068 was purchased from Selleckchem. Glucose-free DMEM was purchased from Gibco Company (USA).

Gao C., Cai Y., Zhang X., Huang H., Wang J., Wang Y., Tong X., Wang J, & Wu J. (2015). Ischemic Preconditioning Mediates Neuroprotection against Ischemia in Mouse Hippocampal CA1 Neurons by Inducing Autophagy. PLoS ONE, 10(9), e0137146.

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Cell Line Culture Protocols

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All the cell lines, including OVCAR83 and SKOV3, were obtained from ATCC. OVCAR83, SKOV3, A549, T47D, and AGS were cultured in 1,640 medium, and Hela and LN229 were cultured in DMEM medium, supplemented with 10% FBS (Gibco, Invitrogen Life Technologies, Carlsbad, CA, USA) in 5% CO₂ at 37°C. Glucose-free DMEM and dialyzed fetal bovine serum were obtained from Gibco, and fructose was purchased from Sigma. In this study, Glucose medium was added 25 mM glucose in Glucose-free DMEM, and fructose medium was added 25 mM fructose in Glucose-free DMEM.13 (link)

Jin C., Gong X, & Shang Y. (2019). GLUT5 increases fructose utilization in ovarian cancer. OncoTargets and therapy, 12, 5425-5436.

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Metabolic Profiling of Cultured Cells

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Glucose uptake was measured using the Glucose Uptake Cell-Based Assay Kit (Cayman) according to the manufacturer’s protocol. Following incubation with fluorescent glucose analog 2-NBDG in glucose-free DMEM (GIBCO) at 37°C for 1 hour and cell preparation according to the manufacturer’s instruction, emission at 535 nm was measured using a fluorescent confocal microscope (Nikon Eclipse Ni-U) and fluorescent intensity quantified in Fiji (NIH). Conditioned media was collected from cells following 48h proliferation in pyruvate-free, 10mM glucose DMEM in order to quantify extracellular lactate normalized to cell count using the L-Lactate Assay Kit I (Eton). Cellular NADPH and NADP+ levels of cells seeded on white 96-well plates in pyruvate-free 10 mM glucose DMEM and lysed in 1% dodecyltrimethylammonium bromide were assayed using the NADP/NADPH-Glo Assay Kit (Promega) according to manufacturer instruction and raw luminescence normalized to cell count measured by the TC-20 automated cell counter (BioRad). GSH/GSSG ratios were measured in cells seeded in pyruvate-free 10mM glucose DMEM using the GSH/GSSG-Glo Assay (Promega) as specified by manufacturer protocol and normalized to cell count.

Hsieh M.H., Choe J.H., Gadhvi J., Kim Y.J., Arguez M.A., Palmer M., Gerold H., Nowak C., Do H., Mazambani S., Knighton J.K., Cha M., Goodwin J., Kang M.K., Jeong J.Y., Lee S.Y., Faubert B., Xuan Z., Abel E.D., Scafoglio C., Shackelford D.B., Minna J.D., Singh P.K., Shulaev V., Bleris L., Hoyt K., Kim J., Inoue M., DeBerardinis R.J., Kim T.H, & Kim J.W. (2019). p63 and SOX2 Dictate Glucose Reliance and Metabolic Vulnerabilities in Squamous Cell Carcinomas. Cell reports, 28(7), 1860-1878.e9.

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In Vitro Model of Ischemic-Reperfusion Injury

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OGD/R, a well-established in vitro model, mimics the conditions of ischemic/reperfusion damage. Primary cortical neuronal cells were seeded at a density of 1×10⁵ cells/mL and bEND.3, BV-2, and RAW 264.7 cells were seeded in 6-well plates at a density of 2×10⁵ cells/well. First, the cells were incubated in glucose-free DMEM (GIBCO) at 37℃ and saturated with a humidified atmosphere of 1% O₂, 94% nitrogen, and 5% CO₂ for OGD. The test formulation was added to the complete medium for preparation. After OGD, the cells were rinsed with PBS three times and subsequently incubated in complete culture medium in the presence of 95% air and 5% CO₂ for reperfusion. Control cells were subjected to the same washing and medium changes but always maintained in complete culture medium under conditions of 95% air and 5% CO₂ at 37℃.

Jin W., Wu Y., Chen N., Wang Q., Wang Y., Li Y., Li S., Han X., Yang E., Tong F., Wu J., Yuan X, & Kang C. (2021). Early administration of MPC-n(IVIg) selectively accumulates in ischemic areas to protect inflammation-induced brain damage from ischemic stroke. Theranostics, 11(17), 8197-8217.

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