OVCAR-8 cells were obtained from Marcus Peter (Northwestern University, Chicago, USA, RRID: CVCL_IM65), TYK-nu cells from Gottfried Konecny (Ronald Reagan UCLA Medical Center, RRID:CVCL_1776), OVCAR-4 cells from Charles River Fredrick National Laboratory for Cancer Research (RRID:CVCL_1627), and PEO-1 cells from Scott Kaufmann (Mayo Clinic, Rochester, MN, RRID:CVCL_2686). OVCAR-8 cells were grown in DMEM (10-013-CV) (Corning) with 10% Fetal Bovine Serum (35-010-CV, FBS) (Fisher Scientific), 1% MEM nonessential amino acids (25-025-CI) (Corning) and 1% MEM Vitamins (25-020-CI) (Corning). TYK-nu cells were grown in MEM (10-022-CV) (Corning) with 10% FBS, 1% MEM nonessential amino acids (25-025-CI) (Corning) and 1% MEM Vitamins (25-020-CI) (Corning). OVCAR-4 cells were grown in RPMI with 10% Fetal Bovine Serum (35-010-CV, FBS) (Fisher Scientific). PEO-1 cells were grown in DMEM (10-013-CV) (Corning) with 10% Fetal Bovine Serum (35-010-CV, FBS) (Fisher Scientific), 1% MEM nonessential amino acids (25-025-CI) (Corning) and 1% MEM Vitamins (25-020-CI) (Corning). Cells were grown in 5% CO2 incubator at 37°C. All cells were mycoplasma negative and their identity verified regularly by short tandem repeat profiling (IDEXX) (OVCAR-8 and TYK-nu Feb 2020, OVCAR-4 June 2019, PEO-1 July 2019). Cells were passaged 2-10 times after unfreezing prior to performing experiments.
Dmem 10 013 cv
Manufactured by Corning
Sourced in United States
DMEM (10-013-CV) is a basal medium used for the growth and maintenance of a variety of cell types in cell culture applications. It provides an optimized balance of nutrients, salts, and other components to support cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Panc 1, by American Type Culture Collection (2 mentions)
Mia paca 2, by American Type Culture Collection (2 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Bovine calf serum (bcs), by American Type Culture Collection (1 mentions)
Pen strep, by American Type Culture Collection (1 mentions)
Dmem 10 017 cv, by Corning (1 mentions)
L glutamine, by Carl Roth (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Corning (1 mentions)
Pen strep, by Lonza (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Tauroursodeoxycholic acid tudca, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Bxpc 3, by American Type Culture Collection (1 mentions)
Aspc 1, by American Type Culture Collection (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Hyclone rpmi 1640 medium, by GE Healthcare (1 mentions)
17 protocols using dmem 10 013 cv
1
Cell Culture Protocols for Ovarian Cancer
2
Bone Marrow-Derived Macrophage Generation
3
Culturing Muller and R28 Retinal Cell Lines
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Muller (ENW001, Kerafast, Boston, MA) and R28 (EUR201, Kerafast) cells were cultured in 5% CO2 humidified incubator at 37°C. The culture media for Muller cells consisted of DMEM (10–017-CV, Corning, Corning, NY) supplemented with final concentrations of 1% Pen-Strep (17-6020E, Lonza, Basel, Switzerland), 10% fetal bovine serum (16140–071, Gibco, Waltham, MA), and additional 1% L-glutamine (3772, Carl Roth, Karlsruhe, Germany). The culture media for the retinal cell line R28 consisted of the following: DMEM (10-013-CV, Corning) supplemented with 1× MEM nonessential amino acids (11140-050, Gibco), 1% Pen-Strep, 10% bovine calf serum (30–2030, ATCC, Manassas, VA), sodium bicarbonate (15 mL of 7.5 w/v %, 7412-12, Mallinckrodt Chemicals, St. Louis, MO), and L-glutamine (5 mL of 200 mM stock, 3772, Carl Roth). We used 1× PBS to dilute Trypsin-EDTA (25-053-CI, Corning) 1:3 for Muller cells. CMF-EDTA was used as the trypsin dilutant for R28 cells. The recipe for CMF-EDTA was followed as outlined by the R28 care document from Kerafast.
4
Pancreatic Cancer Cell Culture and Reagents
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The human pancreatic cancer cell lines PANC-1 (CRL-14690), AsPC-1 (CRL-1682), MIA PaCa-2 (CRL-1420), and BxPC-3 (CRL-1687) were purchased from American Type Culture Collection, and DAN-G was obtained from German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The cells were grown in a 5% CO2 humidified atmosphere in DMEM (10-013-CV, Corning, NY) + 10% FBS (16000-044, Life technologies, Carlsbad, CA, USA), RPMI1640 (10-040-CV, Corning) + 10%FBS. MiaPaCa was cultured in DMEM + 10% FBS + 2.5% Horse serum (R55075, Life technologies). 1% Penicillin-Streptomycin (15140-122, Life technologies) was added to the various culture media.
Non-targeting SiRNA (SIC001) and SiRNA against USP10 (SASI_Hs01_00213007; SASI_Hs01_00213008) were obtained from Sigma-Aldirch. Cycloheximide (C7698), Puromycin (P8833), and Tauroursodeoxycholic acid (TUDCA) (580549) were obtained from Sigma-Aldrich.
Non-targeting SiRNA (SIC001) and SiRNA against USP10 (SASI_Hs01_00213007; SASI_Hs01_00213008) were obtained from Sigma-Aldirch. Cycloheximide (C7698), Puromycin (P8833), and Tauroursodeoxycholic acid (TUDCA) (580549) were obtained from Sigma-Aldrich.
5
Culturing Human Ovarian Cancer Cell Lines
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The human ovarian cancer cell lines were obtained from the American Type Culture Collection (ATCC) and the University of Texas MD Anderson Cancer Center Characterized Cell Line Core Facility. Cell lines were routinely identified via short tandem repeat DNA profiling carried out by the Characterized Cell Line Core Facility at MD Anderson. Primary FTE cells were a gift from J. Liu from the Department of Pathology at MD Anderson. For all cell lines, mycoplasma testing was done using the ATCC PCR Universal Mycoplasma Detection Kit (30-1012K). OVCAR-8 cells were cultured in HyClone RPMI 1640 medium (SH30027.01, GE Healthcare Life Sciences) supplemented with 15% fetal bovine serum (FBS) (Sigma-Aldrich) and 0.2% gentamicin (50146970, Thermo Fisher Scientific). OVCAR-5 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; 10-013-CV; Corning) supplemented with 10% FBS and 0.2% gentamicin. FTE cells were cultured in medium 199 with MCDB 105 (1:1) with 10% FBS and 0.2% gentamicin. All cells were grown in humidified incubators kept at 37°C with 5% CO2.
6
Generating Fibroblasts and HEK293T Cells with POT1 Variants
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Patient-derived skin fibroblasts were obtained from a skin punch biopsy and grown in NUT.MIX.F-12(HAM) w/Glutamax (HAMF-12), supplemented with 10% FBS and penicillin/streptomycin (15140122; Gibco). They were cultured in DMEM (10-013-CV; Corning) supplemented with 1× penicillin/streptomycin (15140122; Gibco) and 10% heat-inactivated FBS (S1620; Biowest) at 37°C and 5% CO2. HEK293T were selected with shRNAs targeting POT1 as in our previous publication (Rice et al., 2017 (link)). These cells were then introduced with Flag-POT1(WT) or FlagPOT1(L259S) using pLU iBLAST vectors. Cells were selected using growth medium supplemented with 5 μg/ml blasticidin S and 2 μg/ml puromycin.
7
Dual-Luciferase Reporter Assay for PPARγ/RXRα Activity
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The reporter assay was performed as described elsewhere (55 (link), 56 (link)). On day 0, HEK293T cells were set up for experiments in 0.5 mL of DMEM (10-013-CV; Corning) supplemented with 10% FBS (16000044; Thermo Fisher Scientific) at a density of 30,000 cells per well in 24-well plates. On day 1, cells were cotransfected with 0.125 ng of pTK-Renilla luciferase, 12.5 ng of pLE1-firefly luciferase or pLE2-firefly luciferase, 162.7 ng of pCMV-PPARγ, and 162.7 ng of pCMV-RXRα. Fugene 6 was used as the transfection agent. For each transfection, the total amount of DNA was adjusted to 338 ng per dish by the addition of pcDNA mock vector. On day 2, the cells were treated with 1 μM rosiglitazone (Sigma) in DMSO, 1 μM 9-cis-Retinoic acid (Sigma–Aldrich) in DMSO, or DMSO alone. On day 3, the cells were washed with PBS, after which luciferase activity was read on a CLARIOstar (BMG Labtech) using the Dual-Luciferase Reporter Assay System (Promega). The amount of LE1 (or LE2) luciferase activity in each dish was normalized to the amount of Renilla luciferase activity in the same dish. A relative luciferase activity of 1 represents the normalized luciferase value in dishes transfected with pcDNA mock vector with DMSO treatment. All values are the average of duplicate assays.
8
Comparative Analysis of Laryngeal Cancer Cell Lines
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Human laryngeal squamous cell carcinoma cell lines, UM-SCC-12 (Black patient derived; RRID: CVCL_7717) and UM-SCC-10A (White patient derived; RRID: CVCL_7713) were authenticated via short tandem repeat typing and further genetically characterized (33 (link), 34 (link)) prior to purchase from the University of Michigan Head and Neck cell line repository. Both cell lines were age, sex, grade and stage matched (see Supplementary Table 1
9
Cell Signaling Pathway Profiling in Cancer Cells
10
Cas9-expressing HeLa and HEK293T cell culture
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HeLa cells that stably express Cas9 protein27 (link) and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, 10–013-CV, Corning, Tewksbury, MA, USA) with 1% penicillin/streptomycin and 10% foetal bovine serum (FBS, Lanzhou Bailing Biotechnology Co., Ltd. Lanzhou, China). All the cells were maintained at 37 °C under 5% CO2. For transfection, 2 × 105 HeLa cells or 4 × 105 HEK293T cells were seeded on 6-well plates and transfected with X-tremeGENE HP (06366546001, Roche, Mannheim, German) according to the supplier’s protocols.
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