The HEK293T (293T) and N2A cells were cultured in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, D5796) with 10% fetal bovine serum and penicillin-streptomycin at 37°C in 5% CO2/95% air. The SH-SY5Y cells were cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium/F-12 Ham (Sigma-Aldrich, D8437) with 10% fetal bovine serum and penicillin-streptomycin at 37°C in 5% CO2/95% air. The 293T cells were transfected with Polyethylenimine “Max” (Polysciences, Inc.). The SH-SY5Y and N2A cells were transfected with Lipofectamine 2000 (Life Technologies, 11668).
Dulbecco s modified eagle s medium f 12 ham
Manufactured by Merck Group
Sourced in United States
Dulbecco's Modified Eagle's Medium/F-12 Ham is a widely used cell culture medium formulation. It is a mixture of nutrients and growth factors that supports the in vitro cultivation of various cell types.
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Lab products found in correlation
Penicillin streptomycin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine max, by Polysciences (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Pyridoxine, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Iowa 1t, by American Type Culture Collection (1 mentions)
Hs 578bst, by Shanghai Cell Bank (1 mentions)
Mda mb 231, by Shanghai Cell Bank (1 mentions)
Mcf 7, by Shanghai Cell Bank (1 mentions)
Distilled water, by Merck Group (1 mentions)
Triton x 100 x100, by Merck Group (1 mentions)
Fecl3 6h2o 1 1 diphenyl 2 picrylhydrazyl dpph, by Merck Group (1 mentions)
6 hydroxy 2 5 7 8 tetramethylchroman 2 carboxylic acid trolox, by Merck Group (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
5 protocols using dulbecco s modified eagle s medium f 12 ham
1
Cell Culture and Transfection Protocol
2
Porcine Jejunal Epithelial Cell Culture Protocol
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The porcine jejunal epithelial IPEC-J2 cell line was used throughout the experiment (kindly provided by Nguyen Lien Thi Minh and Kerstin Skovgaard, Technical University of Denmark, National Veterinary Institute, Denmark). IPEC-J2 is a nontransformed small intestinal cell line developed from a neonatal, unsuckled piglet (<1 day old), maintained as a culture (Berschneider 1989 ) and characterized as a swine-specific in vitro infection model (Schierack et al. 2006 (link)). The cells were grown in Dulbecco's modified Eagle's medium/F-12 Ham containing 0.12% sodium bicarbonate, 15 mM HEPES, pyridoxine and l -glutamine (Sigma Aldrich, St. Louis, Missouri), supplemented with an antibiotic mixture (penicillin, 100 U/mL, and streptomycin, 100 μg/mL), 0.5 mmol/L sodium pyruvate, and 5% fetal bovine serum (Sigma Aldrich), and maintained in an atmosphere of 5% CO2 at 37°C. Cells were passaged every 3–4 days (by seeding at 1:3 ratio). Medium was changed every other day. During experimental periods with bacterial incubation, cell culture medium was replaced with medium containing no antibiotics at least 12 h prior to treatment. In order to keep the cell phenotype stable and consistent (Hubatsch et al. 2007 (link)), cells within 10 passages (passage number 95–105) were used in all experiments.
3
Culturing Breast Cancer and Healthy Cell Lines
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IBC cell line IOWA-1T (ATCC® CRL-3292™) was purchased from the ATCC cell bank. Breast cancer cell lines MDA-MB-231, T47D, and MCF-7 and healthy mammary fibroblasts Hs578Bst were purchased from the Shanghai Cell Bank. Cells were cultured in 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium/F-12 HAM (Sigma-Aldrich Chemical Company, St. Louis, MO, USA) supplemented with 50 units/ml penicillin-streptomycin (Sigma-Aldrich Chemical Company, St. Louis, MO, USA) and 10% fetal bovine serum (FBS; Sigma-Aldrich Chemical Company, St. Louis, MO, USA).
4
Antioxidant Evaluation of Plant Extracts
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HPLC grade acetonitrile, methanol and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Standards for HPLC analysis (phenolic acids: rosmarinic acid, chlorogenic acid, and protocatechuic acid and flavonoids: rutin and isoquercetin) were purchased from Fluka Chemie. All reagents were of analytical grade. Distilled water and ethanol were purchased from Sigma-Aldrich. Trolox (6-hydroxy-2,5,7,8,-tetramethyl-chroman-2-carboxylic acid); and FeCl3·6H2O; 1,1-diphenyl-2-picrylhydrazyl (DPPH) were from Sigma Aldrich. 2,4,6-Trispyridyl-s-triazine (TPTZ) was purchased from Fluka Chemie (Buchs, Switzerland). Ethanol, acetic acid, ammonium hydroxide solution, hydrochloric acid, sodium acetate, and sodium carbonate were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland). Dulbecco’s Modified Eagle’s Medium High Glucose (4500 mg/L), Dulbecco’s Modified Eagle’s Medium F12 HAM, Triton X-100 (X100), penicillin-streptomycin solution, trypsin-EDTA solution, phosphate-buffered saline PBS, MTT reagent, DMSO were purchased from Sigma-Aldrich.
5
Isolation and Culture of Primary Human Osteoblasts and Dermal Microvascular Endothelial Cells
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Human primary osteoblasts (pOB) were obtained from healthy cranial bone obtained as excess tissue during surgery. Cells were isolated from bone fragments according to an established protocol 22 (link) and were cultivated in Dulbecco´s Modified Eagle´s Medium F-12 Ham (Sigma-Aldrich, St. Louis, USA) supplemented with 10% FCS and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, USA) at 37°C in a humidified atmosphere. Primary osteoblasts were utilized up to passage 5. Human dermal microvascular endothelial cells were isolated from healthy tissue remnants obtained during lip operations and according to an established protocol. 23 (link) HDMECs were cultivated in T-25 cell culture flasks previously coated with gelatin (0.02%) in Endothelial Cell Growth Medium MV containing 1% penicillin/streptomycin.
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