Human cervical cancer cell line HeLa used for this study was obtained from the American Type Culture Collection (ATCC) and cells were cultured in DMEM‐F12 (1:1) medium (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% foetal bovine serum (FBS) at 37°C in 5% CO2 humidified incubator. For siRNA transfection HeLa cells (2.0 × 105) were plated in a 6‐well dish, in antibiotic‐free growth medium supplemented with 10% FBS and cultured until 70–90% confluence. Specific siRNA for E7 HPV18 gene was designed and synthesized by Ambion (Thermo Fisher Scientific Inc.) with the following sequences: sense: 5′‐GGAAGAAAACGAUGAAAUAtt‐3′; antisense: 5′‐UAUUUCAUCGUUUUCUUCCtc‐3′. HeLa cells were transfected with siRNA‐E7HPV18 (concentration 75 nM) and siRNA control using Lipofectamine® 2000 Transfection Reagent (Thermo Fisher Scientific Inc.) in Opti‐MEM™ I Reduced Serum Media (Gibco™, Thermo Fisher Scientific Inc.) according to manufacturer's recommendations. Transfection was carried out for 48 hrs and afterwards transfected cells and controls (cells cultured in transfection medium as well as untreated cells) were harvested for further studies. Transfection efficiency was evaluated according to manufacturer instructions 25 by quantifying E7 HPV18 mRNA expression levels in transfected cells and computed to be 75.4%.
Dmem f12 1 1 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China
DMEM/F12 (1:1) medium is a cell culture medium commonly used for the in vitro cultivation of a variety of cell types. It is a formulation consisting of Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture in a 1:1 ratio. This medium is designed to support the growth and maintenance of diverse cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (43 mentions)
Penicillin, by Thermo Fisher Scientific (18 mentions)
Streptomycin, by Thermo Fisher Scientific (18 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (10 mentions)
Hydrocortisone, by Merck Group (9 mentions)
Horse serum, by Thermo Fisher Scientific (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Trypsin, by Thermo Fisher Scientific (7 mentions)
Rpmi 1640, by Thermo Fisher Scientific (6 mentions)
Dmem medium, by Thermo Fisher Scientific (6 mentions)
Axio observer z1, by Zeiss (6 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Insulin, by Merck Group (4 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by Merck Group (4 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (4 mentions)
Colo678, by Leibniz Institute DSMZ (4 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (3 mentions)
Mycoalert plus mycoplasma detection kit, by Lonza (3 mentions)
108 protocols using dmem f12 1 1 medium
1
Gene Silencing of HPV18 E7 in HeLa Cells
2
Cloning and Transfection of Drosophila Odorant Receptor
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Drosophila melanogaster Orco was cloned into pcDNA3.1(−) expression vector as previously described (Mukunda et al., 2014 (link)). HEK cells (DSMZ no. ACC 305) were purchased from the Leibniz Institute DSMZ GmbH (Braunschweig, Germany) and grown in DMEM/F12 1:1 medium (Gibco, Life Technologies, Grand Island, NY, USA) supplied with 10% Fetal Bovine Serum at 37°C and 5% CO2. HEK293 cells were electroporated with 1.6 μg Or83b-pcDNA3.1(−) using an Amaxa 4D-Nucleofector (Lonza GmbH, Cologne, Germany) with the SF Cell Line 4D-Nucleoefector X Kit (Lonza GmbH, Cologne, Germany). After electroporation, cells were cultured on poly-L-lysine (0.01%, Sigma-Aldrich, Steinheim, Germany) coated coverslips at a density of ~3 × 105 cells per well (24 well plates). For experiments cells were exposed to normal bath solution (in mM: NaCl, 135; KCl, 5; MgCl2, 1; CaCl2, 1; HEPES, 10; d-glucose, 10; pH = 7.4; osmolarity 295 mOsmol/l).
3
Comparative Analysis of HNSCC Cell Lines
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Seven human HNSCC cell lines (WSU-HN4, WSU-HN6, WSU-HN30, SCC-4, SCC-9, SCC-25 and CAL-27), a lung cancer cell line (A549) and a cervical cancer cell line (HeLa) were used in this study, and the Research Resource Identifiers (RRIDs) are listed in Additional file 1 : Table S1. The WSU-HN4 (HN4), WSU-HN6 (HN6), and WSU-HN30 (HN30) cells were kindly provided by the University of Maryland Dental School, USA, and the A549 and HeLa cells were purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China. These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, NY), as were the CAL-27 cells (purchased from the American Type Culture Collection, Manassas, VA). The SCC-4, SCC-9 and SCC-25 cells (also from the American Type Culture Collection) were cultured in DMEM/F12 (1:1) medium (Gibco-BRL). The media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco-BRL), penicillin (100 units/mL), and streptomycin (100 μg/mL). The cells were cultured at 37 °C in a humidified 5% CO2 atmosphere. In addition, normal oral epithelial cells were primary cultured in keratinocyte serum-free medium (KSF; Gibco-BRL) with 0.2 ng/mL recombinant epidermal growth factor (rEGF; Invitrogen, Carlsbad, CA, USA).
4
Cell Culture Protocol for Key Cell Lines
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K562 cells were grown in RPMI 1640 (GIBCO) with 25 mM HEPES, 2 mM l -glutamine, 2 g/L NaHCO3 and supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mM l -glutamine. HEK293T cells were grown in Dulbecco’s modified eagle medium (DMEM, GIBCO) with 25 mM d -glucose, 3.7 g/L NaHCO3, 4 mM l -glutamine and supplemented with 10% (v/v) FBS, 100 units/mL penicillin, 100 μg/mL streptomycin. HeLa and H358 cells were grown in the same media as HEK293T cells with the additional supplementation of 2 mM l -glutamine. RPE1 cells were grown in DMEM:F12 (1:1) medium (GIBCO) supplemented with 10% (v/v) FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. K562, HeLa, and RPE-1 cells are derived from female patients/donors. H358 cells are derived from a male patient. HEK293T are derived from a female fetus. All cell lines were grown at 37°C. All cell lines were periodically tested for Mycoplasma contamination using the MycoAlert Plus Mycoplasma detection kit (Lonza).
5
Berberine Modulates Autophagy in Cells
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Berberine was purchased from J&K SCIENTIFIC LTD (China). The RPMI-1640 medium, Dulbecco,s modified Eagle,s (DMEM) medium, DMEM / F-12 1:1 medium and fetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NY). MTT and monodansylcadaverin (MDC) were obtained from Sigma (St. Louis, MO). Trizol, PrimeScript RT Master Mix and SYBR green PCR master mix were from Takara (Shiga, Japan). GRP78 antibody was obtained from Abcam (Cambridge, UK). Antibodies for LC3, p62, AMPK, p-AMPKT183/172 and ATF6 were obtained from Proteintech (Chicago, USA). GAPDH, GRP78, Ub, Beclin1 and chloroquine were purchased from Bioworld Technology (Minneapolis, MN). VPS34 antibody was obtained from Cell Signaling Technology (Danvers, MA). FITC- and TRITC- secondary antibodies were obtained from Invitrogen (Carlsbad, CA). Bafilomycin and 3-methyladenine of autophagy inhibitors were purchased from Cayman Chemical (MI, USA). MG-132 (proteasome inhibitor) and cycloheximide (protein synthesis inhibitor) were obtained from Sigma (St. Louis, USA). Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein standard was obtained from Thermo Scientific. BCA protein assay kit was purchased from Beyotime.
6
Breast Cancer Cell Line Culture and Manipulation
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MCF-7 (ATCC® HTB-22™) human breast cancer cells were grown in DMEM, high glucose, pyruvate (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher). MCF-12A (ATCC® CRL-10782™) human breast epithelial cells were grown in DMEM/F12 (1:1) medium (Gibco), supplemented with 10% horse serum (Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher), 2.5 mg/ml insulin (Invitrogen), 150 μg/ml cholera enterotoxin (Sigma-Aldrich), 2.5 mg/ml hydrocortisone (Sigma-Aldrich) and 20 ng/ml epidermal growth factor (Sigma-Aldrich). Bag-1 knockout MCF-7 cells were generated using CRISPR-Cas9 system [9 (link)]. Cells were maintained at 37°C, 5% CO2 in a humidified incubator. 60–70% confluent cells were transfected with plasmids by using IN-fect in vitro transfection reagent (iNtRON Biotechnology) according to the manufacturer’s protocol. Cells were lysed 48 hours after plasmid transfection for protein purification or immunoblotting assays.
7
Maintenance of MCF10A, MCFDCIS, and HEK293T cell lines
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MCF10A (RRID:CVCL_0598) and MCFDCIS (RRID:CVCL_5552) cell lines [32 (link)] were maintained in DMEM/F12 (1:1) medium (Gibco, Waltham, MA, USA, 11039-021) with 5% horse serum, 20 µg/mL epidermal growth factor (EGF), 100 µg/mL hydrocortisone, 10 µg/mL insulin, and 100 ng/mL cholera toxin. HEK293T cells were maintained in DMEM medium (Gibco, 11995-065) with 10% fetal bovine serum (FBS). All cells were cultured at 37 °C with 5% CO2.
8
Modulating miR-206 Expression in Nasopharyngeal Carcinoma Cells
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CNE-1 cells were obtained from Institute of Virology, Chinese Academy of Preventive Medicine; NP-69 (the SV40 large T immortalized nasopharyngeal epithelial cell line) and CNE-2 cells were purchased from the Shanghai Institute of Cell Biology (Shanghai, P.R. China). The EBV+ C666-1 cells were from the cell bank of Xiangya Central Laboratory (Central South University, Changsha, P.R. China). Cells were maintained in RPMI-1640 or DMEM/F12 (1:1) medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing streptomycin, penicillin, and supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific) and cultured at 37°C in a humidified incubator with 5% CO2. For functional analysis, C666-1 cells grown to 80%–90% confluence were transfected with the miR-206 mimic, the miR-206 inhibitor, or scrambled miRNA as a control (Genepharma Company, Suzhou, P.R. China) using Lipofectamine RNAimax (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Cells were harvested 48–72 h after transfection for further experiments. Lentiviral transduction of C666-1 cells was carried out with lentivirus carrying miR-206 precursor and its control according to the manufacturer’s protocol (Genechem Company, Shanghai, P.R. China). Stable miR-206-overexpressing cells and the control cells were then selected using puromycin.
9
Culturing the A6 Cell Line for ENaC Studies
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The A6 cell line is derived from distal nephron segments of Xenopus laevis and serves as an appropriate cell model which has been extensively used for studying ENaC. A6 cells were purchased from the American Type Culture Collection (Rockville, MD, United States) and grown in medium consisting of three parts DMEM/F-12 (1: 1) medium (Gibco, United States) and one part H2O, with 15 mM NaHCO3 (total Na+ = 101 mM), 2 mM L-glutamine, 10% fetal bovine serum (Invitrogen, United States), 25 units/ml penicillin, and 25 units/ml streptomycin. A6 cells were cultured in plastic flasks in the presence of 1 μM aldosterone at 26°C and 4% CO2. After the cells reached 70% confluence, they were subcultured on the polyester membranes of Transwell inserts (Corning Costar Co., United States) for confocal microscopy analysis. To allow them to be fully polarized, cells were cultured for at least 2 to 3 weeks before performing the experiments (Wang et al., 2018 (link)).
10
Establishing Thyroid Cancer Spheroids
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Tumor and normal thyroid tissues were digested in DMEM/2 mg/ml collagenase type I (Sigma-Aldrich, Saint Louis, MO, USA) at 37°C for 2 h. Cells were seeded at 5×105 cells and grown in serum-free medium: DMEM/F12 1:1 medium (Gibco-Thermo Fisher Scientific, Waltham, Massachusetts, USA) containing 20 ng/ml human basic fibroblast growth factor-bFGF (PeproTech EC, London, UK), 20 ng/ml human epidermal growth factor-EGF (PeproTech EC, London, UK), B27 (1:50 dilution, Gibco-Invitrogen, Carlsbad, CA, USA), antibiotics and Fungizone (Gibco-Thermo Fisher Scientific Waltham, Massachusetts, USA) in ultra-low attachment 6 well plates (Corning Incorporated Life Science, Tewksbury, MA, USA). The wells of the ultra-low attachment plates are coated with polystyrene, an inert substrate, which blocks cell attachment and induces cells in suspension to aggregate into visible spheroids. The cells were incubated in a 37°C, 5% CO2 incubator and floating primary aggregates of cells with spheroid-like structure appeared after 7 days, and fresh medium was added to wells weekly. Spheroids/well were counted using a Leica DMD108 digital microimaging instrument (Leica Microsystems, Milan, Italy) and the spheroids diameter was calculated using AxioVision software rel. 4.8 (Oberkochen, Germany).
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