DMEM was obtained from Life Technologies. Unless otherwise noted, all reagents were obtained from Sigma. The composition of Kreb’s solution used during imaging experiments was as follows: 121 mM Na+, 4.9 mM K+, 25 mM NaCO3–, 1.2 mM Mg2+, 2.5 mM CaCl2, 1.2 mM NaHPO3–, 11 mM d -glucose, and 1 mM pyruvate. Kreb’s solution was titrated to pH 7.4 with NaOH. 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (18:1 LPA; Avanti Polar Lipids, Inc.) was dissolved in 50% ethanol (vol/vol) according to the manufacturer’s instructions, and care was taken to avoid surpassing its critical micelle concentration. AM966 (Cayman Chemical Company; catalog 22048) was dissolved in DMSO to a final stock concentration of 50 mM. Glial metabolism was inhibited with the drug sodium fluoroacetate (FA, 5 mM dissolved in DMEM) for 2 hours and 1 hour in Ca2+ imaging and colonic motor complexes, respectively. Nitric oxide synthase (NOS) was inhibited by l -NAME (Nω-nitro-l-arginine methyl ester, Cayman) at a final concentration of 100 μM in DMEM.
Dulbecco modified eagle medium (dmem)
Manufactured by Cayman Chemical
Sourced in United States
DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium commonly used in laboratories. It provides essential nutrients and growth factors to support the in vitro cultivation of various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Atorvastatin, by Cayman Chemical (2 mentions)
Ay9944, by Cayman Chemical (2 mentions)
Simvastatin, by Cayman Chemical (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Mk886, by Cayman Chemical (1 mentions)
Axiovert 40c microscope, by Zeiss (1 mentions)
D5796, by Merck Group (1 mentions)
Cx 5461, by Cayman Chemical (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Eve automatic cell counter, by NanoEnTek (1 mentions)
L name, by Cayman Chemical (1 mentions)
Nω nitro l arginine methyl ester, by Cayman Chemical (1 mentions)
Ki16425, by Cayman Chemical (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
9 protocols using dulbecco modified eagle medium (dmem)
1
Imaging and Motor Complexes in Colonic Tissue
2
Cholesterol Synthesis Inhibition in Cell Lines
3
Epigenetic Drug Treatments on HeLa Cells
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Human HeLa cells were maintained in DMEM (Corning) with 10% fetal bovine serum (FBS; HyClone) and 1% 100× penicillin/streptomycin (Corning). The cells were incubated at 37°C and 5% CO2 for no more than 30 generations, and were passaged every 2–4 d. Experiments on captured chromosomes used cells that were allowed to recover 1–3 d before capture. Cells were freely cycling and not treated with drugs designed to affect or synchronize the cell cycle.
For epigenetic drug treatments, the cells were plated as above in drug-free DMEM and allowed to recover for ∼8 h, and then treated with 2 mM VPA (Sigma), 50 nM TSA (Sigma), or 2 µM MS (Cayman Chemicals), all dissolved in DMEM. Chromosomes were then captured from the cells (see below) 16–24 h after treatment for VPA and TSA, or 40–48 h for MS treatments.
For epigenetic drug treatments, the cells were plated as above in drug-free DMEM and allowed to recover for ∼8 h, and then treated with 2 mM VPA (Sigma), 50 nM TSA (Sigma), or 2 µM MS (Cayman Chemicals), all dissolved in DMEM. Chromosomes were then captured from the cells (see below) 16–24 h after treatment for VPA and TSA, or 40–48 h for MS treatments.
4
Analyzing PPARα Regulation in Kidney Cells
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The mouse kidney tubular cell line MM55.K (ATCC ref. CRL-6436) was maintained in DMEM (Gibco, #11965092) supplemented with 10% fetal bovine serum (FBS), at 37 °C, 5% CO2 atmosphere and split with trypsin/EDTA solution. Cells were plated onto 6-well plates (3 × 105 cells/well) and, after 24 h of culture, treated with gemfibrozil (100 µM), MK-886 (25 µM, Cayman Chemical, #10133), a PPARα antagonist, and/or metformin (50 µM) in DMEM containing 10% FBS. After 24 h in culture, cells were briefly washed with PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4). RNA extraction, cDNA synthesis, and Real-Time PCR were then conducted.
5
Differentiation and Proliferation of MO3.13 Oligodendrocytes
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The human oligodendrocytic cell line MO3.13 was purchased from Cedarlane (Burlington, ON, Canada). Cells were cultured according to the manufacturer’s protocol in DMEM (D5796, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), 100 μg/ml streptomycin, and 100 U/mL penicillin (both from Sigma-Aldrich, St. Louis, MO, USA) in 5% CO2 at 37 °C. The medium was changed every 2–3 days, and cells were passaged when confluent. To induce differentiation, MO3.13 cells were incubated in the presence of 100 nM PMA (Sigma-Aldrich, St. Louis, MO, USA) or in the presence of 200 nM Pol I inhibitor, CX-5461 (Cayman Chemical, Ann Arbor, MI, USA) in a medium containing DMEM supplemented with 1% FBS. The medium was exchanged every second day, and cell morphology was analyzed daily. Cell morphology was examined using an Axiovert 40C microscope (Carl Zeiss, Jena, Germany) equipped with an A-Plan 10×/0.25 Ph1-objective.
Proliferation of MO3.13 cells was assessed by cell counting, using an EVE automatic cell counter (NanoEnTek, Seoul, Korea). Cells were plated onto a 12-well plate at the density of 150,000 cells per well. After 24 h, cells were treated with PMA (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 100 nM or left untreated (control). Cells were cultured for additional 72 h, then trypsinized and counted.
Proliferation of MO3.13 cells was assessed by cell counting, using an EVE automatic cell counter (NanoEnTek, Seoul, Korea). Cells were plated onto a 12-well plate at the density of 150,000 cells per well. After 24 h, cells were treated with PMA (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 100 nM or left untreated (control). Cells were cultured for additional 72 h, then trypsinized and counted.
6
Cholesterol Synthesis Inhibition in Cell Lines
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HEK293T (parental and hCLTAEN-Tq2) and human SK-MEL-2 (hCLTAEN-Tq2 hDNM2EN-eGFP, as previously described; Scott et al., 2018 (link)) were maintained in DMEM (GIBCO, 4.5 g/L glucose, 110 mg/L pyruvate), supplemented with 10% (v/v) FBS (Hyclone), and 1,000 U/mL penicillin/streptomycin. For inhibition of cholesterol synthesis, cells were rinsed twice in serum-free DMEM and cultured under cholesterol deplete conditions in 7.5% delipidated serum (LPDS) for 48 h with small molecule inhibitors AY9944 (2.5 μM, Cayman Chemical; DHCR7 inhibitor), U18666A (20 nM, Cayman Chemical; DHCR24 inhibitor), Atorvastatin (1 μM, Cayman Chemical), or Simvastatin (Cayman Chemical), or at concentrations as otherwise specified. Acute sterol depletion was achieved by 1 h incubation with 5 mM MβCD (Alfa Aesar, 1303.31 g/mol) at 37°C. De-identified fibroblasts cultured from skin punch biopsies from Smith-Lemli-Opitz subjects (kind gift from Dr. Forbes Porter, NICHD) were maintained in DMEM (GIBCO, 4.5 g/L glucose, 110 mg/L pyruvate), supplemented with 15% (v/v) FBS (Hyclone), and 1,000 U/mL penicillin/streptomycin. SLOS fibroblasts were rinsed twice in serum-free DMEM and cultured for 10 days in 7.5% (v/v) LPDS to induce biochemical profiles. SLOS fibroblast cells lines were described previously (Krakowiak et al., 2000 (link); Wassif et al., 1998 (link)).
7
Mouse CCL11 Secretion ELISA
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In total, 3.5 × 106 FC1245 PDAC cells were seeded in 10-cm plates with DMEM (Thermo Scientific, 11965126) containing 10% VWR Seradigm FBS (VWR, 97068-085) and 1 mM sodium pyruvate (Thermo Scientific, 11360070). For pharmacological treatments, cells were washed with PBS and treated for 24 h in serum-free DMEM with 10 μM Ki16425 (Cayman, 10012659), H2L5186303 (Cayman, 14663) or HA130 (Echelon Bioscience, B-0701). The drugs were reconstituted in DMSO. A sandwich immunoluminometric assay was used to measure the level of mouse CCL11 secreted 24 h after conditioned medium was added (mouse CCL11 Quantikine ELISA kit; R&D Systems, MME00). A Cytiva 28932358 Ultracel 3-kDA filter unit was used to concentrate the conditioned medium samples before ELISA.
8
Comprehensive Reagents for Cellular Assays
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N-acetylcysteine (NAC, A7250), cycloheximide (CHX, 5.08739), dichlorofluorescein diacetate (DCF-DA) (35845), Torin1 (475991), ferrostatin-1 (Ferr-1, SML0583), deferoxamine (DFO, D9533), and MG132 (M7449) were obtained from Sigma-Aldrich (Missouri, USA); SB203580 (S1076), bafilomycin A1 (S1413), and compound C (S7306) were obtained from Selleck Chemicals (USA); STF-31 (4484) was obtained from TOCRIS (USA); ZnPPIX (14483) was purchased from Cayman Chemical; DMEM, RPMI 1640 medium, FBS, amino acids (50X), β-mercaptoethanol, penicillin, and streptomycin were purchased from Gibco (Utah, USA); DMEM (amino acid-free) was purchased from Genetimes Technology (Shanghai, China); the Cell Counting Kit-8 (CCK-8) (K009) was purchased from ZETA LIFE (CA, USA); vitamin C (VC) (ST1434) and puromycin dihydrochloride (ST551) were purchased from Beyotime Biotechnology (Shanghai, China); YF 488-Annexin V/PI Apoptosis Detection Kit (Y6002M) was purchased from US Everbright (Suzhou, China); and TRIzol reagent, PrimeScript RT reagent Kit (RR047A), and TB Green quantitative real-time quantitative polymerase chain reaction (qRT-PCR) kit (RR820A) were purchased from Takara (Dalian, China).
9
Extracellular Vesicle Isolation from iMSCs
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DMEM (Gibco, Waltham, MA, USA) supplemented with 15% FBS was used for iMSC culture. EV-depleted FBS was prepared as previously described [35 (link)]. Cells from passages 6–7 were collected and seeded at a density of 10,000 cells/cm2 in a 150 mm culture dish (SPL, Pocheon-si, Korea). The next day, cells were treated with or without lanifibranor (Cayman, Ann Arbor, M, USA) for 24 h, after which the medium was replaced with DMEM supplemented with EV-depleted FBS and cultured for an additional 24 h. The supernatant was centrifuged for 10 min at 300×g, and the supernatant was transferred to a new tubes, which were then centrifuged for 20 min at 2000×g. This step was followed for another round of centrifugation at 10,000g for 80 min. The supernatant was passed through a 0.2-µm vacuum filter (Merck Millipore, Burlington, MA, USA). Finally, the EVs were isolated by ultracentrifugation at 100,000×g for 80 min, after which the pellet was washed with PBS again (Beckman Coulter, CA, USA). EV pellets were redissolved in EV-free PBS.
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