Glucose free dmem medium

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Glucose-free DMEM medium is a cell culture medium formulation that does not contain glucose. It is designed for the in vitro cultivation of various cell types that may require a glucose-free environment.

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DMEM medium (4294 citations) Glucose-free DMEM (135 citations) Glucose-free medium (20 citations) DMEM-glucose (1 citations)

9 protocols using glucose free dmem medium

Engineered iPSCs for Ttn Mutation Studies

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Patient-specific iPSCs harboring heterozygous A-band TTNtv mutations as well as the wild types were produced from cryopreserved T-cells using STEMCCA lentivirus as described previously (Hinson et al., 2015 (link)). iPSCs were screened for copy number variants and virtual karyotyping using Illumina HumanOmniExpress-12v1 arrays. iPSCs were maintained in complete mTeSR1 medium (Stem Cell) and differentiated to the CM lineage in RPMI 1640 medium (Gibco) supplemented with B27 minus insulin (ThermoFisher) by sequential targeting of the WNT pathway - activating WNT pathway using 12μM of CHIR99021 (Tocris) in Day1 and inhibiting WNT pathway using 5μM of IWP4 (Tocris) in Day3 and Day4. CMs were isolated after showing spontaneous beating (usually between Day9 to Day14) using metabolic selection by adding 4mM of DL-lactate (Sigma) in glucose free DMEM medium (ThermoFisher) for two days and CM purity was determined by FACS analysis (>90% Troponin T+). Following selection, CMs were maintained and assayed in RPMI 1640 medium supplemented with B27 (ThermoFisher) and studied on Day30 post initiation of differentiation. Endogenously tagged GFP-paxillin expressing iPSCs were provided by the Allen Institute for Cell Science Human iPSC Collection and cultured as described above.

Chopra A., Kutys M.L., Zhang K., Polacheck W.J., Sheng C.C., Luu R.J., Eyckmans J., Hinson J.T., Seidman J.G., Seidman C.E, & Chen C.S. (2018). Force generation via β-cardiac myosin, titin, and α-actinin drives cardiac sarcomere assembly from cell-matrix adhesions. Developmental cell, 44(1), 87-96.e5.

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Hypoxia and Glucose Starvation in Hepatic Cell Lines

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HepG2, Huh7, and MHCC97H cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Gibco, USA), and L02 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and antibodies. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. Hypoxia experiments were performed using a hypoxic chamber (Stemcell Technologies) with 1% O₂ for 12 h. For glucose starvation, cells were incubated in glucose-free DMEM medium (Thermo Fisher Scientific) for 12 h. Polyamines rescue experiment was performed using 10 μM polyamines mix (Macklin) including the solution of putrescine, spermidine, and spermine as previously described [21 ]. During incubation with polyamines, 1 mM aminoguanidine was routinely added in the medium as an inhibitor of bovine serum amine oxidase to prevent oxidation of extracellular polyamines to toxic products.

Zhang H., Li X., Liu Z., Lin Z., Huang K., Wang Y., Chen Y., Liao L., Wu L., Xie Z., Hou J., Zhang X, & Liu H. (2024). Elevated expression of HIGD1A drives hepatocellular carcinoma progression by regulating polyamine metabolism through c-Myc–ODC1 nexus. Cancer & Metabolism, 12, 7.

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Luciferase Assay for Mitochondrial Toxicity

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HeLa cells were cultivated in glucose-free DMEM medium (Thermo Fisher Scientific) supplemented with 10% FCS, 50 U/ml penicillin, 50 mg/ml streptomycin, and 10 mM galactose (Sigma). Plasmid transfection was performed as previously described and culture media was replaced with serum-free glucose-free DMEM medium 6 h after transfection. Transfected cells were harvested 24 h after transfection and split in 96-well plate at a density of 5 × 10⁴ cells/well, and luciferase activity was measured 24 h later using Mitochondrial ToxGlo Assay (Promega) following manufacturer's recommendations. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment at 20 μM for 90 min was used as positive control.

Caval V., Suspène R., Khalfi P., Gaillard J., Caignard G., Vitour D., Roingeard P., Vartanian J.P, & Wain-Hobson S. (2021). Frame-shifted APOBEC3A encodes two alternative proapoptotic proteins that target the mitochondrial network. The Journal of Biological Chemistry, 297(3), 101081.

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Oxygen and Glucose Deprivation Neuroinflammation

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BV-2 microglial cell line, purchased from China Infrastructure of Cell Line Resources (Beijing, China), was grown in DMEM F12 medium (Gibco, USA) supplemented with 10% FBS (Invitrogen, USA), and 1% penicillin–streptomycin (Invitrogen, USA). oxygen and glucose deprivation/reoxygenation (OGD/R) was conducted according to a previously established protocol [17 (link)]. Normal cell culture medium was removed and changed by glucose-free DMEM medium (Gibco, USA). Then, cells were incubated in an oxygen-free chamber equipped with AnaeroPack-Anaero (MGC, Japan) at 37 °C for 2 h. Finally, the cells were returned to normal incubator and incubated with the initial culture medium for reoxygenation.

Hong Y., Lyu J., Zhu L., Wang X., Peng M., Chen X., Deng Q., Gao J., Yuan Z., Wang D., Xu G, & Xu M. (2022). High-frequency repetitive transcranial magnetic stimulation (rTMS) protects against ischemic stroke by inhibiting M1 microglia polarization through let-7b-5p/HMGA2/NF-κB signaling pathway. BMC Neuroscience, 23, 49.

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Oxygen and Glucose Deprivation/Reoxygenation in Primary Cortical Neurons

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Primary cortical neurons were isolated from fetal rats as previously reported (Bailey and Lahiri, 2006 (link)). The cells were cultured with neurobasal medium (Gibco; Thermo Fisher Scientific, Waltham, MA, United States) supplemented with 2% B27 (Gibco, United States) and 1% glutamine (Gibco, United States) in an incubator with 5% CO₂ at 37°C. The medium was half-replaced every 3 days. The neurons were used to establish oxygen and a glucose deprivation/reoxygenation (OGD/R) model after 7 days. Primary neurons were cultured in a glucose-free DMEM medium (Gibco, United States) in an atmosphere of 5% CO₂ and 1% oxygen and 94% N₂ for half an hour. After OGD operation, the cells were cultured in original medium for 24 h at 37°C in a humidified incubator with 5% CO₂.

Fei H., Xiang P., Luo W., Tan X., Gu C., Liu M., Chen M., Wang Q, & Yang J. (2021). CTRP1 Attenuates Cerebral Ischemia/Reperfusion Injury via the PERK Signaling Pathway. Frontiers in Cell and Developmental Biology, 9, 700854.

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Metabolic Flux Analysis of Cancer Cells

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TPL was purchased from Adamas Reagent Co., Ltd. (Shanghai, Chian). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). Other reagents were obtained from the following companies: methanol (Kermel, Tianjin, China), chloroform, hexane, and pyridine (Aladdin, Shanghai, China), [¹³C-U₆] Glucose, [¹³C-U₅] glutamine (Cambridge, Boston, MA, USA), 2% methoxylamine hydrochloride, N-tert-butyldimethylsily-N-methyltrifluoroacetamide (MTBSTFA), tert-butyldimethylchlorosilane (tBDMS; Sigma, St. Louis, MO, USA), Glucose-free DMEM medium, and glutamine-free RPMI-1640 medium (Gibco, Waltham, MA, USA). Glucose, oligomycin, 2-DG, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and rotenone-antimycin A (Agilent Technologies, Santa Clara, CA, USA). L-glutamine and pyruvate (Thermo Fisher Scientific, Waltham, MA, USA).

Ye S., Xu P., Huang M., Chen X., Zeng S., Wang Q., Chen J., Li K., Gao W., Liu R., Liu J., Shao Y., Zhang H., Xu Y., Zhang Q., Zhong Z., Wei Z., Wang J., Hao B., Huang W, & Liu Q. (2020). The heterocyclic compound Tempol inhibits the growth of cancer cells by interfering with glutamine metabolism. Cell Death & Disease, 11(5), 312.

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Modeling Cerebral Ischemia in BV2 Microglia

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The BV2 microglia (Concord Cell Resource Center, China) growth in 90% high glucose Dulbecco’s Modified Eagle Medium (DMEM) medium supplemented with 10% fetal calf serum (Gibco, United States), 1% penicillin-streptomycin (Gibco, United States), and cells were cultured at 37°C in humidified 5% CO2/95% air for 7–10 days prior to experimentation. The liquid was changed every other day.
BV2 microglia were exposed to OGD for establishing an in vitro model of cerebral ischemia. Before exposure, cultured cells were pretreated with Tanshinone IIA (10 μM) and then the cells seeded in glucose free DMEM medium (Gibco, United States) transferred into an anoxic device (Billups Rothenberg, United States), and flushed of a 95% N₂ and 5% CO₂ gas mixture for 4 h. Control group cells grow normally in the incubator.

Song Z., Feng J., Zhang Q., Deng S., Yu D., Zhang Y, & Li T. (2021). Tanshinone IIA Protects Against Cerebral Ischemia Reperfusion Injury by Regulating Microglial Activation and Polarization via NF-κB Pathway. Frontiers in Pharmacology, 12, 641848.

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Ischemia-Reperfusion Injury in Cardiomyocytes

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Cell cultures were subjected to three hours OGD followed by one hour of reoxygenation. To simulate ischemia, glucose-free DMEM medium (Gibco, USA) was pre-equilibrated in 95% N₂ and 5% CO₂ at 37 °C. Oxygenated medium was removed from the cardiac myocyte cultures, and the N₂-preequilibrated glucose-free DMEM was promptly added. Cultures were immediately placed in a hypoxia container and exposed to 95% N₂ and 5% CO₂ for three hours at 37 °C. For reoxygenation, the glucose-free DMEM was replaced with high-glucose DMEM, and the cardiomyocytes were cultivated in 21% O₂–5% CO₂–74% room air for 1 hour at 37 °C. Cardiac myocytes unexposed to OGD/R served as the normoxic control.

Pei W., Hu H., Liu F., Xiao B., Zuo Y, & Cui W. (2018). C-reactive protein aggravates myocardial ischemia/reperfusion injury through activation of extracellular-signal-regulated kinase 1/2. Journal of Geriatric Cardiology : JGC, 15(7), 492-503.

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Lipid Profiling and KRAS Inhibition

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DL-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) was purchased from Enzo Biochem (# BML-SL210-0010) and dissolved in DMSO. AMG510, a KRASG12C inhibitor, was purchased from Selleck Chemicals (S8830). Brain PtdSer (#840032 C), PIP2 (#840046X), C17:0 Gb3 (#860699), GM3 (#860058), C18 GlcCer (#860547), C18:1 LacCer (#860590), C18:1 GalCer (#860596) and brain SM4 (#131305) were purchased from Avanti Polar Lipids, GM2 from Sigma (G8397). Cell culture medium and glucose-free DMEM medium were purchased from Gibco (Cat#11966025). FBS was purchased from GIBCO. Puromycin was purchased from Thermo Fisher Scientific (BP2956-100). Anti-GFP antibodies used for immunogold labeling were made in house, and anti-GM3 antibodies used for immunogold and immunofluorescent labeling were purchased from Amsbio LLC (GMR6).

Liu J., van der Hoeven R., Kattan W.E., Chang J.T., Montufar-Solis D., Chen W., Wong M., Zhou Y., Lebrilla C.B, & Hancock J.F. (2023). Glycolysis regulates KRAS plasma membrane localization and function through defined glycosphingolipids. Nature Communications, 14, 465.

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