HepG2 cells were seeded in DMEM (containing 4500 mg/L glucose and phenol red) (Life Science, Seoul, Korea) with 10% FBS and 1% PEST at a density of 106 cells/well in a 6-well plate for 24 h, and the medium was changed to assay medium (DMEM without glucose, phenol red and FBS), to which either C3G (10 and 50 μM) was added or metformin was added as a positive control, and treated with or without the compound C (AMPK inhibitor) for another 24 h. Cells were washed 2 times with 1× PBS; then, DMEM containing 20 mM sodium lactate, 2 mM sodium pyruvate, 2 mM L-glutamine and 15 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) was added to the cells; and 100 μM of 8-(4-Chlorophenylthio)adenosine 3′,5′-cyclic monophosphate sodium salt (8-CPT-cAMP) was added as the control for 6 h. The culture medium was collected, and glucose concentrations were determined using an automated clinical chemistry analyzer (Cobas111, Roche, Basel, Switzerland) according to the manufacturer’s instructions.
Dulbecco modified eagle medium (dmem)
Manufactured by Roche
Sourced in Germany, Switzerland
DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium commonly used for the growth and maintenance of various types of mammalian cells. It provides essential nutrients, vitamins, and salts required for cell proliferation and survival. DMEM is a widely used and well-established medium in the field of cell biology and biotechnology.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (11 mentions)
Penicillin streptomycin solution, by Solarbio (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Dnase 1, by Roche (5 mentions)
Fetal bovine serum (fbs), by Roche (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (3 mentions)
Fibronectin, by Thermo Fisher Scientific (2 mentions)
Extracellular matrix, by Merck Group (2 mentions)
Bsa fraction 5, by Roche (2 mentions)
Fibronectin, by Roche (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Nicotinamide, by Merck Group (2 mentions)
Transferrin, by Merck Group (2 mentions)
Sodium selenite, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (2 mentions)
Collagenase a, by Merck Group (2 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (2 mentions)
Percoll, by GE Healthcare (2 mentions)
Wst 1, by Roche (2 mentions)
31 protocols using dulbecco modified eagle medium (dmem)
1
Glucose Metabolism in HepG2 Cells
2
Culturing Insulin-Producing EndoC-βH1 Cells
3
Assessing Alpha Mangostin Cytotoxicity on HepG2 and A549 Cells
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Liver-derived HepG2 and lung-derived A549 cells were used to assess the mammalian cell cytotoxicity of alpha mangostin using previously described methods (Kim et al., 2018b (link); Peng et al., 2021 (link)). The cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, MD, USA) and 1% penicillin/streptomycin (Gibco, MD, USA) and maintained at 37°C in 5% CO2. Cells were harvested and resuspended in a fresh medium, and 100 μl were distributed in a 96-well plate at 1 × 106 cells/well. Alpha mangostin were serially diluted in serum, antibiotic-free DMEM added to the monolayer of the cells, and the plates were incubated at 37°C in 5% CO2 for 24 h. At 4 h, before the end of the incubation period, 10 μl of 2-(4-iodophenyl)-3- (4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium (WST-1) solution (Roche, Mannheim, Germany) was added to each well. WST-1 reduction was monitored at 450 nm using a using SpectraMax M2 Multi-mode Microplate Reader (Molecular Devices, CA, USA). Assays were performed in triplicate, and the percentage of cell survival was calculated. The lethal dose (LD50) is considered the concentration of compounds responsible for killing 50% of cells (Musa et al., 2010 (link)).
4
Isolation of Brain Microvascular Endothelial Cells
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Brain MVEC (bMVEC) were isolated by incorporating and slightly modifying previously described methods [35 (link),36 (link),37 (link),38 (link)]. Brains of 6–8 wk old male C57BL6/J mice (4–6 animals per isolate), were excised and stored in serum-free DMEM (Sigma, St. Louis, MO, USA) on ice before surgical removal of the olfactory bulbs, cerebellum and mid-brain white matter. The remaining cortical tissue was rolled on sterile filter paper and subsequently digested in DMEM containing 2 mg/mL collagenase A (Roche, Basel, Switzerland) and 10 µg/mL DNase I (Roche) at 37 °C for 1 h, with gentle rotation. Digested tissue was pelleted at 290× g and resuspended in DMEM containing 20% BSA (Sigma) (w/v), then myelin fraction was separated by centrifugation at 1000× g for 10 min. The cell pellet was resuspended and filtered through a 70 µM nylon mesh and collected following centrifugation at 290× g. Cells were further digested in 2 mg/mL collagenase/dispase (Roche) and 10 µg/mL DNase I at 37 °C for 30 min. Following one wash in DMEM at 290× g, bMVEC were selected in medium supplemented with 4 µg/mL puromycin dihydrochloride (Sigma) [39 (link),40 (link)] for the first 4 days.
5
Isolation of Mononuclear Cells from PBL
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Mononuclear cells were isolated from PBL by ficoll density gradient centrifugation as previously described35 (link). NP samples were minced using a scalpel, placed in a 50 ml tube with 8 ml DMEM + Liberase TL 125 ug/mL (Roche) + DNAse I 100 ug/ml (SIGMA) + antibiotic/antimycotic (GIBCO), incubated at 37 °C for 45 min and vortexed at low speed each 10 min. After the incubation the suspension was filtered through a sterile 70 um mesh.
6
GPI-anchored Fluorescent Protein Variants
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U2OS cells were maintained in DMEM (Lonza) supplemented with 10% fetal bovine serum (FBS, Gibco) in a humidified incubator at 37 °C, supplied with 5% CO2. A humanized cDNA encoding sGFPori fused to the GPI-anchoring domain of CD1414 (link) was used as template to generate different GPI-sFP variants by site-directed mutagenesis (Quickchange, Agilent Technologies) with appropriate primers. All the constructs were verified by DNA sequencing. Cells transiently transfected with the different GPI-sFP fusions (XtremeGENE HP, Roche) were imaged after incubation with 37 μM of M3 peptide in the DMEM + 10% FBS culture media for 12 hours. Cells were briefly rinsed with HBSS buffer (Corning) and imaged in the same buffer at 37 °C. Confocal fluorescence images were acquired on a Nikon C2 inverted confocal microscope equipped with a 488 nm excitation laser and a 525DF50 nm emission filter for imaging complemented GPI-sGFP variants and with a 515 nm excitation laser and a 542DF27 nm emission filter for imaging complemented GPI-sYFP variants.
7
Evaluating AMP Cytotoxicity in HepG2 Cells
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We used liver-derived HepG2 cells to evaluate AMP cytotoxicity behavior in mammalian cells based on an established protocol [15 (link)]. We maintained the HepG2 cells at 37 °C in 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Gaithersburg, MD, USA) and 1% penicillin/streptomycin (Gibco, Gaithersburg, MD, USA). Next, we harvested and resuspended the cells in a fresh medium and distributed 50 µL (1 × 106 cells) in a 96-well plate containing 50 µL of serially diluted AMPs in serum and antibiotic-free DMEM and incubated the plates at 37 °C in 5% CO2 for 24 h. Before the end of the incubation period (at the 20th hour), we added 10 µL of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) (Roche, Mannheim, Germany) to each well and monitored the reduction of WST-1 at 450 nm using a SpectraMax i3x (Molecular Devices, San Jose, CA, USA). We performed the assays in triplicates and calculated the percentage of cell survival.
8
Culturing and Transfecting HEK-293 Cells Expressing CaSR
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HEK-293 cells stably expressing the human CaSR (HEK-CaSR cells) or the hCaSR/rmGluR1/rmGluR1 (HEK-C/G/G) chimeric receptor were cultured in DMEM (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) as described previously (Mun et al., 2004 (link); Szekely et al., 2009 (link)). Cells were grown on 15-mm diameter glass coverslips in 24-well plates for microfluorimetry experiments.
For experiments with transiently expressing CaSR constructs, when the cells had reached 85%–95% confluency they were transfected with XtremeGENE HP transfection reagent (Roche, Germany): briefly, 0.5 μg samples of WT or mutant DNA and 3 μL of transfection reagent were diluted with 100 μL of DMEM and allowed to complex at RT for 15 min. The transfection solution was added to the cell cultures to a final concentration of 9.1% (v/v).
For experiments with transiently expressing CaSR constructs, when the cells had reached 85%–95% confluency they were transfected with XtremeGENE HP transfection reagent (Roche, Germany): briefly, 0.5 μg samples of WT or mutant DNA and 3 μL of transfection reagent were diluted with 100 μL of DMEM and allowed to complex at RT for 15 min. The transfection solution was added to the cell cultures to a final concentration of 9.1% (v/v).
9
Isolation and Culture of Muscle Progenitor Cells
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Hind limb muscles were homogenized in DMEM (Gibco) using a Miltenyi gentleMACS dissociator. Upon decanting, triturated muscle was digested in Collagenase A (Sigma) and DNAse I (Roche) in DMEM, filtered through 80 μm and 20 μm nylon mesh and pelleted. Cells were resuspended in PBS containing also 0.5% bovine serum albumin 2 mM EDTA, and incubated with CD31 and CD45 microbeads (Miltenyi) before being subjected to magnetic separation in MACS columns. The unretained CD31-CD45- fraction was then incubated with Sca-1-microbeads and processed as described above. After quantification with DAPI and propidium iodide, CD31-CD45-Sca-1 cells were seeded in 8-well chamber slides (LabTek) at 3000 live cells/well in F12-Ham medium supplemented with 1% penicillin/streptomycin, 20% Fetal Calf Serum (Gibco) and 5 ng/mL murine FGFb. Cells were cultured for eight days in the presence or in the absence of curcumin.
10
Akt Signaling Pathway Activation
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