Mouse embryonic fibroblast (MEF) cell line was derived from Abcg2−/− knockout (KO) mice. The Abcg2-KO mice were obtained from the Sorrentino lab.2 (link) The Abcg2-KO MEFs were engineered to express human ABCG2 (Uniprot: Q9UNQ0). Cells were cultured at 37°C with 5% of CO2. Cells were grown in Dulbecco’s Modified Essential Medium (D-MEM) (Lonza, Basel, Switzerland) supplemented with 10% of fetal bovine serum (Gibco, Grand Island, NY), penicillin (100 U/mL), streptomycin (100 μg/mL) (Gibco, Grand Island, NY), and l-glutamine (2 mM) (Gibco, Grand Island, NY). The Abcg2-KO MEF cell line was transduced with the retroviruses harboring cDNAs encoding either the mutant ABCG2 N436A, F439A, F439W, or F439Y.
Dulbecco s modified
Manufactured by Lonza
Sourced in Belgium
Dulbecco's Modified Eagle's Medium (DMEM) is a cell culture medium commonly used for the growth and maintenance of various cell lines. It is a modification of the original Eagle's Minimum Essential Medium, with additional components to provide a more complete and balanced nutrient environment for cell growth. DMEM is suitable for use in both standard cell culture and stem cell research applications.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Lonza (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Mcdb 201 medium, by Merck Group (1 mentions)
Trypan blue, by Sartorius (1 mentions)
Penicillin streptomycin solution, by Harvard Bioscience (1 mentions)
L glutamine, by Harvard Bioscience (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Merck Group (1 mentions)
Heparan sulfate, by Merck Group (1 mentions)
Dextran sulfate, by Merck Group (1 mentions)
Dermatan sulfate, by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Chondroitin sulfate, by Merck Group (1 mentions)
7 protocols using dulbecco s modified
1
Engineered ABCG2 Mutant MEFs
2
Adhesion Assay of MSCs and SaOS-2 Cells
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Total 308000 MSCs and 308000 SaOS-2 cells were used. Adhesion assays were carried out on days 3 and 7. Control groups were MSCs and Saos type 2 cells cultured in the appropriate medium without samples. Tests were carried out in triplicate for each material and cell group. The cells were cultured with MSC or SaOS-2 medium, according to cell type. MSC culture medium consisted of 52.8% Dulbecco’s Modified Eagle Medium (DMEM) with 1 g/L glucose (Lonza, Basel, Switzerland), 35.2% MCDB-201 medium (Sigma Aldrich, St. Louis, MO, USA), 10% heat inactivated fetal bovine serum (FBS) (Sigma Aldrich, St. Louis, MO, USA), 1% penicillin/streptomycin solution (Biochrom AG, Berlin, Germany), and 1% l -glutamine (Biochrom AG, Berlin, Germany), while SaOS-2 culture medium consisted of 89% DMEM with 4.5 g/L glucose (Sigma Aldrich), 10% heat inactivated FBS, and 1% penicillin/streptomycin solution. Every three to four days, the medium was replaced. The plates were incubated at 37 °C with relative humidity under an atmosphere of 5% CO2. On the third and seventh days, the wells were washed with PBS (Oxoid, Hampshire, United Kingdom) and 1 mL Trypsin/EDTA (Thermo Fisher, Waltham, MA, USA) was applied. A 50 µL cell suspension was mixed with 50 µL Trypan blue (Biological Industries, Beit HaEmek, Israel) in a centrifuge tube and the cells were counted on Thoma lamina.
3
Cell Culture and Glycosaminoglycan Characterization
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All cells used for this study were cultured at 37°C under 5% CO2. HEK 293T (ATCC: CRL-1573) cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Verviers, Belgium). Jurkat (ATCC: TIB-152) and BHK-21 (CCL-10) cells were grown in Roswell Park Memorial Institute medium (RPMI; Biowest, Nuaille, France). CHO-K1 and pgsA-745 cells were grown in Ham’s F-12 medium (Life technologies, Darmstadt, Germany) and 2 mM glutamine. Media were supplemented with 10% FBS (v/v; PAA, Pasching, Austria) and 5% L-glutamine (200 mM; Lonza, Verviers, Belgium). The glycosaminoglycans chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and dextran sulfate were purchased from Sigma-Aldrich (Taufkirchen, Germany).
4
Cultivation of Intestinal and Immune Cells
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The human intestinal epithelial HT-29 (ATCC® HTB-38™) and the human monocyte THP-1 (ATCC® TIB-202™) cell lines were maintained in an atmosphere containing 5% CO2 at 37°C in the culture media recommended by ATCC. The human mucus-producing intestinal epithelial HT29-16E cell line [22 (link)] were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum (Lonza), 1% l-glutamine (Life Technologies), 200 U penicillin, 50 mg of streptomycin, and 0.25 mg of amphotericin B per litre. In addition, THP-1 monocytes were differentiated into macrophages by treatment with 20 ng/ml phorbol myristate acetate (PMA) for 18 h.
5
Evaluating AgNPs Cytotoxicity on Cell Lines
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The effect of the AgNPs was evaluated by MTT assay, as previous described, on the human cell lines, KMST-6 normal skin fibroblasts, HT-29 and CaCo-2 colon carcinoma cells [80 (link)]. The cells were purchased from ATCC, and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza) supplemented with 10% fetal bovine serum (Gibco, Waltham, Massachusetts, USA) and 1% penicillin-streptomycin cocktail (Lonza) and incubated at 37 °C. The cells were then seeded in 96 well plates at 1 × 105 cell/mL density, 100 µL in each well and incubated for 24 hr. The cells were treated with 0–500 μg/mL of the AgNPs and extracted in triplicates. The cell viability was assessed by adding 10 μL of 5 mg/mL of MTT (Sigma) solution to each well and incubated for 3 hr. Later, the MTT solution was discarded and 100 μL of DMSO was added to each well. The absorbance of the formazan product was measured at 570 nm with a reference at 700 nm using a POLARstar Omega plate reader. The concentration that inhibited 50% cell growth (IC50) was further analyzed by Graphpad Prism 6.0.
6
SARS-CoV-2 Variant Propagation in Cells
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Vero E6 and RAW 264.7 cells were cultured and maintained at 37 °C, 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (Lonza) with 10% FBS (Gibco) and 1% pen-strep (Lonza). The SARS-CoV-2 clinical isolates, hCoV-19/Korea/KDCA119861/2021 (B.1.617.2 lineage, Delta variant, Clade G) and hCoV-19/Korea/KDCA447321/2021 (B.1.1.529 lineage, Omicron variant, Clade GR) obtained from the National Culture Collection for Pathogens (NCCP) in the Korea Disease Control and Prevention Agency (KDCA) were propagated in Vero E6 cells. The titrated viral stocks were stored at −80 °C until further use.
7
Culturing Human Skin Cell Lines
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NHDF and NHEK were isolated as previously described [46 (link)]. NHEK were cultured at 37 °C, 5% CO2 and 20% O2 in low-Calcium, serum-free DermaLife K medium (Lifeline; Carlsbad, CA, USA) and NHDF at 37 °C, 5% CO2 and 5% O2 in high glucose and l -glutamine-containing Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Verviers, Belgium). The DMEM was supplemented with 10% fetal calf serum (FCS; Invitrogen, Darmstadt, Germany) and 1% antibiotics (Lonza) Medium was generally changed every 2–3 days. For subculturing, NHEK were treated with 0.05% EDTA (Serva Electrophoresis GmbH, Heidelberg, Germany) for 5 min and 0.4% trypsin for 2 min, while NHDF were treated with 0.05% trypsin for 5 min only. Mycoplasma and virus contamination was excluded by the Multiplex Cell Contamination Test (Multiplexion) (Heidelberg, Germany).
HEK293 and A431 were a gift from Prof. Herrmann-Lerdon (DKFZ, Heidelberg, Germany), were cultivated in DMEM and passaged once per week.
Human induced pluripotent stem cells (hiPSCs) were produced and kindly provided by Prof. Jochen Utikal (DKFZ, Heidelberg, Germany).
HEK293 and A431 were a gift from Prof. Herrmann-Lerdon (DKFZ, Heidelberg, Germany), were cultivated in DMEM and passaged once per week.
Human induced pluripotent stem cells (hiPSCs) were produced and kindly provided by Prof. Jochen Utikal (DKFZ, Heidelberg, Germany).
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