Human lung cancer cell line A549 (Male), mouse fibroblast cell line L929 (Male), and human embryonic kidney cell line 293T (Female) were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The mouse macrophage cell line RAW264.7 was a generous gift from Xin Lin Laboratory, School of Life Sciences, Tsinghua University, Beijing, China. A549 cells were grown in RPMI-1640 medium (Wisent, Montreal, QC) supplemented with 10% FBS (Wisent, Montreal, QC) and 1% penicillin/streptomycin (Wisent, Montreal, QC). L929 cells were grown in RPMI-1640 medium (Wisent, Montreal, QC) supplemented with 10% heat-inactivated FBS (Wisent, Montreal, QC) and 1% penicillin/streptomycin (Wisent, Montreal, QC). 293T cells were grown in Dulbecco’s modified Eagle’s medium (Wisent, Montreal, QC) supplemented with 10% FBS (Wisent, Montreal, QC) and 1% penicillin/streptomycin (Wisent, Montreal, QC). RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (Wisent, Montreal, QC) supplemented with 10% heat-inactivated FBS (Wisent, Montreal, QC) and 1% penicillin/streptomycin (Wisent, Montreal, QC). The mycoplasma contamination test for all cells was negative.
Dulbecco s modified eagle s medium
Manufactured by Wisent
Sourced in Canada, China, United States
Dulbecco's modified Eagle's medium (DMEM) is a cell culture media formulation that provides essential nutrients for the growth and maintenance of various cell types. It is a commonly used medium in biological research and cell-based applications. DMEM supplies cells with a balance of amino acids, vitamins, salts, and other components necessary for cellular metabolism and proliferation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Wisent (18 mentions)
Penicillin streptomycin, by Wisent (8 mentions)
Rpmi 1640 medium, by Wisent (4 mentions)
Penicillin, by Wisent (4 mentions)
Streptomycin, by Wisent (4 mentions)
Sodium pyruvate, by Wisent (3 mentions)
L glutamine, by Wisent (3 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
Eagle s minimum essential medium, by Wisent (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Wisent (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Iodoacetamide iaa, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Wisent (2 mentions)
Sequencing grade modified trypsin, by Promega (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Heat inactivated fbs, by Wisent (1 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (1 mentions)
36 protocols using dulbecco s modified eagle s medium
1
Cell Culture Conditions for Various Cell Lines
2
Transient Transfection and Knockdown in Melanoma Cells
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A2058 (CRL-11147) and A375 (CRL-1619) melanoma cells were obtained from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (Wisent) supplemented with 10% fetal bovine serum (American Type Culture Collection) in 5% CO2 according to the supplied guidelines. Mycoplasma contamination was tested biweekly. Transient transfections were performed using polyethyleneimine (PEI) as described previously (14 (link)). Briefly, 1.5 μg total plasmid DNA was diluted in 50 μl Opti-MEM, 5 μl of 1 mg/ml PEI was added, and the mixture was incubated for 25 to 30 min at room temperature. The DNA–PEI mix was added to cells in 1 ml of Opti-MEM and left for 5 h under normal culture conditions. At the end of 5 h, the media were replaced with 2 ml of the appropriate culture medium. siRNA-mediated knockdown was performed as previously described (42 (link)) using Dharmafect1 (PerkinElmer) and the following siRNA duplexes: FMNL2 siRNA Duplex1 (IDT; hs.Ri.FMNL2.13.1); FMNL2 siRNA duplex2 (IDThs.Ri.FMNL2.13.2); IRTKS duplex1 (IDT, hs.Ri.BAIAP2L1.13.1); IRTKS duplex2 (IDT, hs.Ri.BAIAP2L1.13.2); and IRSp53 duplex1 (IDT, hs.Ri.BAIAP2.13.1).
3
Establishing LPS-Induced Renal Epithelial Cell Model
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Human renal tubular epithelial cells (HK-2) were obtained from the American Type Culture Collection (Rockville, MD, USA). Dulbecco’s modified Eagle’s medium (Wisent, Shanghai, China) containing 10% (v/v) fetal bovine serum (FBS; Hyclone, South Logan, UT, USA) and 1% penicillin/streptomycin (GIBCO BRL, Grand Island, NY, USA) was used to culture HK-2 cells in a humidified atmosphere containing 5% CO2 at 37°C. To establish an LPS (L4516-1MG; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) stimulation for cell culture, HK-2 cells were incubated with increasing concentrations (0 mg, 0.1 mg, 1.0 mg, 10 mg, and 20 mg) of LPS for 24 h or treated with 1.0 mg LPS for 0 h, 6 h, 12 h, 18 h, and 24 h).
4
Generation and Characterization of WT and NLRX1-KO MEFs
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Murine embryonic fibroblast (MEF) cells were cultured in Dulbecco's modified Eagle's medium (Wisent, Canada) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Wisent) and 1% penicillin/streptomycin. Cells were maintained in 95% air and 5% CO2 at 37 °C. Generation of primary WT and NLRX1-KO MEFs has been described previously (5 (link)). WT and NLRX1-KO MEF transformed cell lines were generated using the SV40 large T antigen. In brief, WT and NLRX1-KO MEFs were transduced with purified SV40 large T antigen LentifectTM Lentiviral Particles (GeneCopoeia) for 24 h, and puromycin was added to select cells that were positive for the SV40 large T antigen. Cells positive for the SV40 large T antigen were verified by measuring the expression of the SV40 large T antigen by quantitative PCR (qPCR). Similarly, WT and NLRX1-KO SV40-Tschopp MEFs were also monitored for SV40 expression and grown in similar conditions as described above.
5
Transfection and Selection of Cas9-expressing Cells
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HEK293T cells were purchased from ATCC and were cultured in Dulbecco’s modified Eagle’s medium (Wisent) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Pen-Strep; GIBCO). 16HBE cells (a generous gift from Dr. D.C. Gruenert, University of California, San Francisco, CA, USA) and CFF-16HBEge W1282X-CFTR cells (from Cystic Fibrosis Foundation) were cultured in Eagle’s minimum essential medium (Wisent) supplemented with 10% fetal bovine serum and 1% Pen-Strep (GIBCO). We seeded 500,000 cells for transfection in 2 mL of media in six-well plates using Lipofectamine 3000 (Thermo Fisher Scientific). Cells were transfected with 3,000 ng of a Cas9 and guide RNA co-expression vector, pSpCas9(BB)-2A-Puro (PX459) V2.0, which was a gift from Feng Zhang (Addgene plasmid #62988). To enrich for transfected cells, we subject 24 h post-transfection cells to 0.9 μg/mL or 2 μg/mL of puromycin, for HBE cells and HEK293T cells, respectively, for 72 h.
6
Culturing and Activating Primary CD4+ T Cells
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First, 293T human embryonic kidney cells (obtained from ATCC, Manassas, VA, USA) were cultured at 37 °C under 5% CO2 in Dulbecco’s modified Eagle’s medium (Wisent) containing 5% fetal bovine serum (VWR, Radnor, PA, USA) and 100 µg/mL of penicillin-streptomycin (Wisent, St. Bruno, QC, Canada). Primary CD4+ T lymphocytes were purified from resting PBMCs by negative selection and activated as previously described [12 (link),13 (link)]. Briefly, PBMC were obtained by leukapheresis. CD4+ T lymphocytes were purified using immunomagnetic beads as per the manufacturer’s instructions (StemCell Technologies, Vancouver, BC, Canada). CD4+ T lymphocytes were activated with phytohemagglutinin-L (PHA-L; 10 µg/mL) for 48 h and then maintained in RPMI 1640 (Gibco, Waltham, MA, USA) complete medium supplemented with rIL-2 (100 U/mL).
7
Infection and Survival of Bacterial Strains in Macrophage Cell Lines
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The THP-1 (ATCC TIB-202) cells were cultivated in RPMI 1640 (Wisent, St-Bruno, QC, Canada) supplemented with 10% heat-inactivated FBS (Wisent), 1 mM sodium pyruvate (Wisent), and 1% MEM non-essential amino acids (Wisent, St-Bruno, QC, Canada). The human monocytes cells were differentiated into macrophages by addition of 10−7 M phorbol 12-myristate 13 acetate (Sigma) for 48 h before the infection. Similarly, the RAW264.7 (ATCC TIB-71) murine macrophages were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM; Wisent, St-Bruno, QC, Canada). The method was adapted to 96-well plates and performed at a MOI of 10 [36 (link)]. To obtain a similar number of intracellular bacteria, a MOI of 10 was used for macrophages to compensate for the phagocytic activity. Briefly, following an overnight growth in LB broth, the strains were added in triplicate. After 30 min, infected cells were washed with PBS, treated with gentamicin (50 ug/mL), and lysed with PBS-DOC 0.1% at 30 min (phagocytosis), and 18 h (survival) post-infection, then, serial dilutions were performed for enumeration of viable colony counts (CFU/mL). Each deletion was tested at least three times in triplicate.
8
Esophageal Squamous Cell Carcinoma Cell Lines
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ESCC cell lines, including TE-1, KYSE-150, and TE-10, were purchased from American Type Culture Collection (ATCC, Manassas, MA, USA). Normal human esophageal epithelial cells (HEEC) and ESCC cells, including the EC-1 and EC-109 cell lines, were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, PR China). The culture conditions were as follows: Dulbecco’s Modified Eagle’s Medium (Wisent Bioproducts, St-Bruno, QC, Canada) supplemented with 10% FBS (Wisent Bioproducts), 100 U/mL penicillin, and 100 µg/mL streptomycin in a moist incubator (stabilized at 5% CO2 and 37°C).
9
Culturing Human Cell Lines for Osteosarcoma Research
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Human osteosarcoma U-2 OS cells (HTB-96, ATCC), BJ fibroblasts (a gift from S. W Lowe) and fibroblasts from progeria patient (HGADFN003, Progeria Foundation) were cultured in Dulbecco's modified Eagle's medium (Wisent, St-Bruno, QC) supplemented with 10% (for U-2 OS and BJ) or 15% (for HGADFN003) fetal bovine serum (FBS) (Wisent). Flavopiridol was from (Selleckchem, http://www.selleckchem.com/ ). PD0332991 and FTI-277 were purchased from Sigma-Aldrich.
10
Culturing Breast Cancer Cell Lines
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Breast adenocarcinoma cell lines MCF-7 and MDA-MB-231 were cultured in Dulbecco's modified Eagle's medium (Wisent, Canada) supplemented with 10% fetal bovine serum (Invitrogen, USA), 50 μg/ml streptomycin and 50 U/ml penicillin. Cells were kept at 37¼C in a humidified incubator with 5% CO2.
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