Dulbecco s minimal essential medium

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Dulbecco's minimal essential medium (DMEM) is a cell culture medium used to support the growth and maintenance of a wide variety of mammalian cell lines. It is a basal medium that provides essential nutrients, vitamins, and amino acids required for cell proliferation.

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Minimal essential medium (177 citations) Dulbecco’s minimal essential medium (DMEM) (30 citations) Dulbecco’s modified minimal essential medium (25 citations) Dulbecco minimal essential medium (DMEM) (8 citations) Dulbecco’s Modified Eagle’s Minimal Essential Medium (8 citations) Dulbecco’s modified minimal essential medium (DMEM), (4 citations) Dulbecco’s modified Eagle’s minimal essential medium (DMEM; (3 citations) Dulbecco’s minimal essential medium/F12 (2 citations) Dulbecco's modified Eagle minimal essential medium (2 citations)

68 protocols using dulbecco s minimal essential medium

Cell Culture of H9C2 Cardiomyocytes

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H9C2 cells, an immortalized cell line derived from fetal rat hearts were purchased from National Cell bank of Iran (NCBI). Cells at a density of 10⁵/cm² were cultured as monolayers in Dulbecco’s Minimal Essential Medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin and streptomycin (Invitrogen). Cells were counted under invert microscopy using improved hemo-cytometer neobar. Culture medium was replaced by fresh medium every two days. Control and test substrates were inserted to the bottom of 24-well tissue culture plates. Before loading cells on the surface of substrates, PBS used in protein coating step was completely removed from wells and 1 ml of the culture media including cells was added to the wells including substrates. Plates were incubated at standard cell culture conditions (temperature 37 °C and CO₂ 5%). Incubated plates were used for evaluation of cell attachment and cell growth and proliferation. For each evaluation, all experiments were done in triplicate.

Behjati M., Moradi I, & Kazemi M. (2015). Application of novel anodized titanium for enhanced recruitment of H9C2 cardiac myoblast. Iranian Journal of Basic Medical Sciences, 18(9), 873-877.

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Aconitase-2 Suppression in A375 Melanoma Cells

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The human melanoma cell line A375 was obtained from ATCC (Manassas, VA, USA). Cells were cultured in Dulbecco’s minimal essential medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 U/mL of penicillin and 100 µg/mL of streptomycin. A375 cells were infected with the lentiviral pLKO.1-shACO2 (TRCN0000056561, Dharmacon, Lafayette, CO, USA) to suppress aconitase-2 or the control pLKO.1 for 3 or 6 days prior to EPR. Lentivirus production and infection procedures were previously described [27 (link),28 (link),29 (link)]. Cells were treated with 10 µM chromate (Sigma, St. Louis, MO, USA) in HBSS (Invitrogen) in a CO₂ incubator at 37 °C for 30 min and were harvested using cell scrapers. Cells were then collected by centrifugation, washed in ice-cold 1× PBS, resuspended in 0.3 mL 1× PBS, loaded into EPR tubes and snap-frozen. Cells were also harvested, counted and lysed in 62.5 mM Tris (pH 6.8) –2% SDS mixed with protease and phosphatase inhibitor cocktail (Sigma). Protein levels of ACO2, ACO1 and actin were determined by Western blot analysis as previously described [27 (link),28 (link),29 (link)].

Antholine W.E., Vasquez-Vivar J., Quirk B.J., Whelan H.T., Wu P.K., Park J.I, & Myers C.R. (2019). Treatment of Cells and Tissues with Chromate Maximizes Mitochondrial 2Fe2S EPR Signals. International Journal of Molecular Sciences, 20(5), 1143.

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Epigenetic Modulation of Human Hepatoma Cells

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Human hepatoma HepG2, HuH7, and JHH1 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing 10 % fetal bovine serum at 37 °C in a humidified atmosphere containing 5 % CO₂. In order to reverse DNA methylation, the cells were treated with 0.5 or 5 μM 5-aza-2′-deoxycytidine (DAC; Sigma-Aldrich, St. Louis, MO, USA) for 72 h. After DAC treatment, the cells were treated with trichostatin A (TSA; Sigma-Aldrich) at 200 nM (HepG2 and HuH7 cells) or 20 nM (JHH1 cells) for 24 h.

Habano W., Kawamura K., Iizuka N., Terashima J., Sugai T, & Ozawa S. (2015). Analysis of DNA methylation landscape reveals the roles of DNA methylation in the regulation of drug metabolizing enzymes. Clinical Epigenetics, 7, 105.

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Culturing FU-sensitive Human PDAC Cells

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The FU-sensitive (FU-S) human PDAC cell line AsPC-1 (ATCC, Rockwell, MD, USA) was maintained in culture as adherent monolayer in Dulbecco’s minimal essential medium (Invitrogen, Karlsruhe, Germany), supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany) and 1% penicillin/streptomycin (Invitrogen) in 5% CO₂ in an humidified atmosphere at 37 °C.

Lee L.D., Pozios I., Liu V., Nachbichler S.B., Böhmer D., Kamphues C., Beyer K., Bruns C.J., Kreis M.E, & Seeliger H. (2022). Thymidine phosphorylase induction by ionizing radiation antagonizes 5-fluorouracil resistance in human ductal pancreatic adenocarcinoma. Radiation and Environmental Biophysics, 61(2), 255-262.

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Pancreatic Cancer Cell Line Characterization

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The human PDAC cell lines L3.6pl and AsPC-1 were used for in vitro and in vivo studies. AsPC-1 cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). L3.6pl is a secondary highly metastatic human pancreatic adenocarcinoma cell line derived from an orthotopic mouse xenograft model [15 ]. All cell lines were maintained in Dulbecco’s Minimal Essential Medium (Invitrogen GmbH, Karlsruhe, Germany), supplemented with 10% fetal bovine serum and 1% penicillin streptomycin and were incubated in a humidified atmosphere of 5% CO₂ at 37 °C.
Raloxifene was purchased from Sigma-Aldrich (Schnelldorf, Germany) and was dissolved in 0.1% dimethyl sulfoxide (DMSO, Carl Roth GmbH & Co. KG, Karlsruhe, Germany). IL-6 was purchased from Invitrogen GmbH (Karlsruhe, Germany) and was dissolved in acetic acid. Anti-STAT3, anti-phosphorylated-STAT3 (Y705) and anti-gp130 antibodies were obtained from Cell Signaling Technology (Frankfurt am Main, Germany).

Pozios I., Seel N.N., Hering N.A., Hartmann L., Liu V., Camaj P., Müller M.H., Lee L.D., Bruns C.J., Kreis M.E, & Seeliger H. (2020). Raloxifene inhibits pancreatic adenocarcinoma growth by interfering with ERβ and IL-6/gp130/STAT3 signaling. Cellular Oncology (Dordrecht), 44(1), 167-177.

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Culturing Schwannoma and HeLa Cell Lines

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The HEI-193 human schwannoma cell line (from D.J. Lim, House Ear Institute, Los Angeles, CA, USA) was established from a NF2-schwannoma patient, immortalized with human papillomavirus E6/E7 genes and grown as described [37 (link)]. The Mouse 08031-9 schwannoma cells (from Dr. Marco Giovannini, Univ. of California, Las Angeles, CA, USA) were grown as described [38 (link)]. All cells were infected with lentivirus encoding Fluc and mCherry for bioluminescence imaging and immunohistochemistry, respectively [12 (link)]. HeLa cells were cultured as monolayers in Dulbecco’s Minimal Essential Medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum and 5% penicillin and streptomycin. Cells were incubated in a 37 °C incubator in an atmosphere of 10% CO₂ in air. HeLa cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Ahmed S.G., Maguire C.A., Cao S.A, & Brenner G.J. (2022). Schwannoma Gene Therapy via Adeno-Associated Viral Vector Delivery of Apoptosis-Associated Speck-like Protein Containing CARD (ASC): Preclinical Efficacy and Safety. International Journal of Molecular Sciences, 23(2), 819.

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TGF-β1 Regulation of Cell Lines

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The HaCaT human keratinocyte cell line, the Caco2 human colon cancer cell line and the HEK293 human embryo kidney cell line (ATCC) were maintained at 37 °C under 5% CO₂ in Dulbecco's minimal essential medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The human colon cancer cell lines HT29 and HCT116 (ATCC) were maintained in McCoy's 5A medium supplemented with 10% fetal calf serum. TGF-β1 (100–21) was purchased from Pepro Tech (Rocky Hill, NJ, USA). Cells were treated with 10 ng/ml TGF-β1 for 20, 24 or 48 h, washed with phosphate-buffered saline, and harvested for downstream assays.

Shen L., Qu X., Ma Y., Zheng J., Chu D., Liu B., Li X., Wang M., Xu C., Liu N., Yao L, & Zhang J. (2014). Tumor suppressor NDRG2 tips the balance of oncogenic TGF-β via EMT inhibition in colorectal cancer. Oncogenesis, 3(2), e86-.

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Transient Transfection of RFP-SND1 in HeLa Cells

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HeLa cells were purchased from the American Type Culture Collection and cultured in Dulbecco's minimal essential medium (Invitrogen Life Technologies, Madrid, Spain) containing 10% fetal bovine serum. A plasmid encoding RFP epitope‐tagged SND1 (RFP‐SND1) was generated as previously described (Gao et al., 2010). Transfections of HeLa cells were performed using L‐polyethyleneimine (Sigma–Aldrich, St Louis, MO), according to the manufacturer's instructions.

Shao J., Gao F., Zhang B., Zhao M., Zhou Y., He J., Ren L., Yao Z., Yang J., Su C, & Gao X. (2017). Aggregation of SND1 in Stress Granules is Associated with the Microtubule Cytoskeleton During Heat Shock Stimulus. Anatomical Record (Hoboken, N.j. : 2007), 300(12), 2192-2199.

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Temperature-dependent ARPE-19 Cell Culture

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The human RPE cell line, ARPE-19, was obtained from the American Type Culture Collection (Manassas, VA, USA). Cultures were seeded with 5 × 10⁵ cells in 35-mm culture dishes with 2 ml of Dulbecco’s minimal essential medium (Invitrogen, Carlsbad, CA, USA) containing antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin sulfate; Invitrogen) and 10% fetal calf serum, and grown at 37°C in an atmosphere of 5% CO₂. Subsequently, medium change was performed every 24 h. At day 2, cells reached confluence. At day 3, medium was changed with 2 ml of Dulbecco’s minimal essential medium containing antibiotics and 1% fetal calf serum. At day 4, medium was changed, and cells were transferred to temperature-adjusted incubators at 37, 35, 33 and 31°C in an atmosphere of 5% CO₂. The ARPE-19 cell samples and conditioned media were collected at 24 h and 48 h after the start of incubation at each adjusted temperature. Conditioned media samples were stored at −80°C until use.

Takeyama M., Yoneda M., Gosho M., Iwaki M, & Zako M. (2015). Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia. Biological Research, 48(1), 42.

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Isolation of Midbrain Astrocyte Cultures

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Astroglial cultures were prepared from the midbrain of E13.5 embryos from time-mated C57BL/6 mice (Harlan Sprague-Dawley; Indianapolis, IN). A modified protocol from Ho and Blum (1997) was used for the preparation of astroglial cultures [19 (link)]. Tissues were incubated in 5 mL KRB containing 20 μL trypsin (Gibco/Invitrogen; Carlsbad, CA) for 5 min at 37°C, centrifuged (3000 × g, 10 min), decanted and triturated in 5 mL of calcium and magnesium-free KRB buffer containing 50 μL DNase (Gibco/Invitrogen). The triturated cells were centrifuged again, decanted and the pellet was resuspended in 20 mL Dulbecco’s minimal essential medium (Gibco/Invitrogen) supplemented with 10% fetal bovine serum (FBS). The cell suspension was filtered through a 70 μm nylon mesh screen (Falcon; Franklin Lakes, NJ). Cells were grown in 5% CO₂ at 37°C. Confluent monolayers were shaken at 37°C for 2 hr at 250 rpm to remove microglia. The purity of the cells was assessed using antibodies directed against MAC-1 and GFAP for identification of microglia and astrocytes, respectively and MAC-1 positive cells represented less than 5% of the total cell population.

Bains M, & Roberts J.L. (2015). Estrogen protects against dopamine neuron toxicity in primary mesencephalic cultures through an indirect P13K/Akt mediated astrocyte pathway. Neuroscience letters, 610, 79-85.

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