The human squamous cell carcinoma cell line FaDu (obtained from ATCC; ATCC® HTB43™) and the murine colon carcinoma CT26.WT (hereinafter referred to as CT26) tumor cell line (obtained from ATCC, Manassas, VA, USA; ATCC® CRL-2638™) were cultured in the ATCC-suggested cell culture mediums. The FaDu cells were cultured in Advanced Dulbecco’s Modified Eagle’s Medium (A-DMEM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and the CT26 cells were cultured in Advanced RPMI-1640 (A-RPMI, Gibco), both supplemented with 2 mM L-glutamine (Gibco), 5% (v/v) fetal bovine serum (FBS, Gibco), GlutaMAX (Gibco) and 1% (v/v) Penicillin–Streptomycin (stock solution, 10,000 U/mL, Gibco). Additionally, HEK-Blue™ IL-12 cells (IL-12 reporter cells) (InvivoGen) were used to determine the biological activity and potency of phIL12 and pmIL12. The HEK-Blue™ IL-12 cells were cultured in A-DMEM (Gibco) supplemented with 2 mM L-glutamine (Gibco), 5% (v/v) FBS (Gibco) and 1% (v/v) Penicillin–Streptomycin (stock solution, 10,000 U/mL, Gibco). After the second passage, the 1× HEK-Blue™ Selection (InvivoGen) was added to the growth medium. The cells were handled according to the supplier’s instructions and cultured in a 5% CO2 humidified incubator at 37 °C. For the experiments, the cells were maintained in monolayers until they reached 70–80% confluence.
A dmem
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
A-DMEM is a basal cell culture medium designed for the growth and maintenance of a variety of mammalian cell types. It provides the necessary nutrients and components to support cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
F 12 ham, by Merck Group (3 mentions)
Human recombinant tnfα, by Cayman Chemical (2 mentions)
Mycoalert mycoplasma detection kit, by Lonza (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Mc3t3 e1 cell, by American Type Culture Collection (2 mentions)
Luciferin, by GoldBio (2 mentions)
Dexamethasone (dex), by Thermo Fisher Scientific (2 mentions)
White 96 well plate, by Greiner (2 mentions)
M200 pro reader, by Tecan (2 mentions)
Multicycle, by Actimetrics (2 mentions)
Ccl 171, by American Type Culture Collection (1 mentions)
Tib 67, by American Type Culture Collection (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Promega (1 mentions)
Htb 43, by American Type Culture Collection (1 mentions)
Hek blue selection, by InvivoGen (1 mentions)
Crl 2638, by American Type Culture Collection (1 mentions)
26 protocols using a dmem
1
Cell Culture Protocols for Tumor Cell Lines
2
Isolation and Culture of Rat Bone Marrow Stromal Cells
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The remaining 10 rats from each group with or without depression were used to isolate BMSCs as previously described.9 (link) Briefly, rats were euthanized and bilateral femurs were removed. The marrow was extruded by flushing with excessive amounts of α-Dulbecco’s Modified Eagle’s Medium (α-DMEM, Gibco, Langley, OK, USA), and the bone marrow aspirated was subjected to density gradient centrifugation to collect the mononuclear cells. Then, these cells were seeded into a 25 cm2 plastic flask and cultured in α-DMEM containing 10% fetal bovine serum (FBS, Gibco) at 37°C in a humidified 5% CO2 atmosphere. After 3 days culture, the nonadherent cells were removed by changing the culture medium, and the adherent cells were further cultured and the medium was refreshed every 2 days. The BMSCs were passaged upon reaching 80%–90% confluence, and cells from the third passage (P3) were used for experiments.
3
Mycobacterial Infection Cell Culture
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Human lung fibroblast cell line MRC-5 (ATCC® CCL-171™) and macrophage-like cell J774A.1 derived from BALB/c mouse tumour (ATCC® TIB-67™) were maintained and expanded in Advanced DMEM (A-DMEM) (Life technologies) supplemented with 5% new-born calf serum and 0.3% (w/v) L-glutamine at 37 °C with 5% CO2 until 80% confluent.
WT M. marinum M (ATCC BAA-535™) and a RD1 knockout with M. marinum M background (a kind gift from Fredric Carlsson, Lund university) were grown in 7H9 medium (BD Difco) supplemented with 0.05% Tween80 and 10% ADS (0.5% albumin, 0.2% dextrose and 0.085% saline) as standing cultures at 30 °C. M. tuberculosis H37Rv and H37Ra were also grown in 7H9 medium supplemented with 0.1% Tween80 and ADS as standing cultures at 37 °C.
WT M. marinum M (ATCC BAA-535™) and a RD1 knockout with M. marinum M background (a kind gift from Fredric Carlsson, Lund university) were grown in 7H9 medium (BD Difco) supplemented with 0.05% Tween80 and 10% ADS (0.5% albumin, 0.2% dextrose and 0.085% saline) as standing cultures at 30 °C. M. tuberculosis H37Rv and H37Ra were also grown in 7H9 medium supplemented with 0.1% Tween80 and ADS as standing cultures at 37 °C.
4
Maintaining Hepatocyte Viability with LPS
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Culturing and Treating Bladder Cancer Cells
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Human urothelial carcinoma cell line RT4 (HTB-2, ATCC, Manassas, VA, USA) was grown in basal media consisting of equal parts of advanced Dulbecco’s Modified Eagle’s Medium (A-DMEM) (Gibco, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (HAM) (Sigma-Aldrich, Merck, Darmstadt, Germany) supplemented with 5% fetal bovine serum (FBS; Gibco, Life Technologies, Carlsbad, CA, USA) and 4 mM GlutaMAX (Gibco, Life technologies, Carlsbad, CA, USA) in 75 cm2 culture flasks. For the experiments, RT4 cells were seeded on coverslips in 6-well plates (for fluorescence microscopy) or 96-well plates without coverslips (for measuring fluorescence intensity) at a seeding density of 1 × 105 cells/cm2 and grown until reaching 80–90% confluence. To mimic a proinflammatory environment, cells were treated with 20 ng/mL human recombinant TNFα (Cayman Chemicals, Ann Arbor, MI, USA) in serum-free basal media for 24 h, as previously described [45 (link),46 (link),47 (link)]. Untreated cells grown in serum-free basal media served as controls.
6
Evaluating Genotoxic Stress Response in Fibroblasts
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Fibroblasts were isolated from skin biopsies of 6 healthy controls and 6 patients carrying the identified APTX mutation. Cells were cultured in Advanced DMEM/F-12 (Dulbecco's Modified Eagle Medium/Ham's F-12) (ADMEM, Gibco) containing 10% fetal bovine serum (FBS) (Gibco), 2mM L-glutamine (Life Technologies), 100 U/ml penicillin and streptomycin (Gibco) and maintained in a humidified incubator under 37°C and 5% CO2. Cells were cultured in 96-well plates at a density of 5000 cells/well. The following day, cells were treated in triplicates with increasing concentrations of mitomycin-C (MMC), methyl-methane sulfonate (MMS) and etoposide for 1 hour, and H2O2 for 30 minutes, under 37°C and 5% CO2. Cells in normal culture media with no treatment served as a control. To assess the effect of these different genotoxic agents on cell survival, we performed MTT assay [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] (Promega, USA) after 48 hours of treatment and the percentage of viable cells was calculated accordingly.
7
Isolation and Culture of Rat Bone Marrow Mesenchymal Stem Cells
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rBM-MSCs were isolated from long bones of 2–4 weeks old Lewis rats by the experimental animal service of the Scientific Park of Barcelona (SEA-PCB). Rats were anesthetized with 5% isoflurane and sacrificed in a CO2 saturated atmosphere [33 (link)]. rBM-MSCs were cultured in aDMEM (Gibco, Barcelona, spain) supplemented with 10% FBS (Sigma, Madrid, Spain), 1% penicillin/streptomycin (100 µg/mL) and 1% L-glutamine (2 mM; Sigma). Passages between 4–6 were used in all experiments. All animal care protocols were approved by the Committee on Ethics and Animal Experiments of the Scientific Park of Barcelona (Permit No. 0006S/13393/2011, 2011).
8
Culturing SV-HUC1 Human Urothelial Cells
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Human normal urothelial cells SV-HUC1 (CRL-9520, ATCC, Manassas, VA, USA) were grown in 75 cm2 cell culture flasks in basal media consisting of equal parts of advanced Dulbecco’s modified Eagle’s medium (A-DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (HAM) (Sigma Aldrich, St. Louis, MO, USA), supplemented with 5% fetal bovine serum (FBS) and 4 mM GlutaMAX (both Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells repeatedly tested negative for mycoplasma infection using MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland). For the experiments, SV-HUC1 cells were seeded into appropriate plates/chambers at a seeding density of 3 × 104 cells/cm2 and grown until reaching 70–80% confluency (approximately 3–4 days) before performing experiments. All experiments were performed in serum-free basal media.
9
Culturing Human Epithelial and Endothelial Cells
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Two lines of epithelial cells were examined in this work, namely, human intestinal epithelial cells (HUIEC) that were previously isolated and characterized by our group [73 (link)] and a line of human umbilical vein endothelial cells (HUVEC) purchased from ATTC. A base culture of cells was maintained in Advanced Dulbecco’s Modified Eagle’s Medium (ADMEM, Gibco, Thermo Scientific, Waltham, MA, USA), supplemented with 5% (wt) fetal bovine serum (FBS, Gibco, Thermo Scientific, Waltham, MA, USA) at 37 °C and 5% CO2.
10
Proinflammatory Stimulation of Urothelial Cells
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Human non-invasive cancer urothelial cells, RT4, isolated from transitional cell papilloma (HTB-2, ATCC, Manassas, VA) were grown in 75 cm2 cell culture flasks in basal media consisting of equal parts of advanced Dulbecco’s modified Eagle’s medium (A-DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and F12 (HAM) (Sigma Aldrich, St. Louis, MO, USA), supplemented with 5% fetal bovine serum (FBS), and 4 mM GlutaMAX (both Gibco, Thermo Fisher Scientific, USA). Cells repeatedly tested negative for mycoplasma infection using MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland). For the experiments, RT4 cells were seeded in 6-well plates with (for IF) or without (for RNA and protein extraction and measurement of protein levels in supernatants) coverslips or 96-well plates (for viability assay) at a seeding density of 5×104 cells/cm2 and grown until reaching 80-90% confluency (approximately 3-4 days). To mimic a proinflammatory environment, cells were treated with 20 ng/ml human recombinant TNFα (Cayman Chemicals, Ann Arbor, MI, USA) for 24 h in serum-free basal media, as previously described (18 (link)–21 (link)). Untreated cells grown in serum-free basal media served as controls.
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