The PC-3 prostate cancer and SKBR-3 breast cancer cell lines were obtained from the Cell Bank resources of the University of Barcelona. Cells were grown in Ham’s F12 medium with 10% fetal bovine serum and were incubated at 37 °C in a humified atmosphere at 5% CO2. The hCMEC/D3 cell line was grown in Endothelial Cell Basal Medium-2 (EBM®-2, from Lonza, Walkersville, MD, USA). Subculture was performed with Trypsin 0.05% (Merck, Madrid, Spain).
Trypsin 0.05
Manufactured by Merck Group
Sourced in Spain
Trypsin 0.05% is a laboratory reagent used in cell culture and related applications. It is a proteolytic enzyme that is used to dissociate adherent cells from a culture surface. Trypsin 0.05% is a concentrated solution that is typically diluted before use.
Automatically generated - may contain errors
Lab products found in correlation
Ebm 2, by Lonza (1 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Transwell insert, by Corning (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Bronchial epithelial growth medium (begm), by Lonza (1 mentions)
Singlequots, by Lonza (1 mentions)
Penicillin streptomycin, by Euroclone (1 mentions)
Cabozantinib, by Cayman Chemical (1 mentions)
F12k kaighn s modification medium, by Thermo Fisher Scientific (1 mentions)
Vandetanib, by Cayman Chemical (1 mentions)
4 protocols using trypsin 0.05
1
Culturing PC-3, SKBR-3, and hCMEC/D3 Cell Lines
2
Cell Line Gene Silencing Experiments
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The different cell lines used for gene silencing experiments were SH-SY5Y, PC-3, MDA-MB-453-WT, HepG2, CHO, and Vero-E6. Cell lines were obtained from the Cell Bank resources of the University of Barcelona. Cells were grown in Ham’s F12 medium supplemented with 10% fetal bovine serum or in RPMI medium supplemented with 7% dialyzed fetal bovine serum for the experiments of plasmid transfection (all from GIBCO, Invitrogen, Barcelona, Spain). Cells were incubated at 37 °C in a humidified 5% CO2 atmosphere. Subculture was performed with Trypsin 0.05% (Merck, Madrid, Spain).
3
Culturing and Differentiating Respiratory Epithelial Cells
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Epithelial cell culture and passage were performed using methods previously described.7 , 16 In brief, samples were separated from the brush head, pelleted, and then cultured on collagen‐coated flasks in bronchial epithelial growth medium (BEGM) (CC‐4175, Lonza, Basel, Switzerland), supplemented with penicillin/streptomycin 100 U/mL (Sigma, Gillingham, UK), incubated at 37°C in a 5% CO2 incubator. Medium was changed every 2 to 3 days. Passage was performed when cells reached 70% to 80% confluence. The cells were removed from the culture surface with trypsin 0.05% (Sigma, Gillingham, UK), neutralized with 10% Fetal Bovine Serum (Sigma, Gillingham, UK), centrifuged, and then placed in a new growth container. Cells were also cultured at air–liquid interface (ALI), for which cells are exposed to air apically and medium basally. In these conditions, respiratory epithelial cells typically differentiate into a pseudostratified respiratory epithelium with ciliated and mucus‐producing cells.17 To achieve this, cells were passaged onto transwell inserts (0.4‐μm pore size, Corning, Corning, NY) and cultured with BEGM medium supplemented with SingleQuots (Lonza, Switzerland) and antibiotics.16 , 18
4
Evaluation of Vandetanib and Cabozantinib in MTC Cell Lines
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Vandetanib (VAN), and cabozantinib (CAB) were provided by Cayman Chemicals (Ann Arbor, MI, USA). Stock solutions (4 mM) were made in 100% dimethyl sulfoxide (DMSO) and diluted with culture media before use. Two human medullary thyroid carcinoma (MTC) cell lines, TT and MZ-CRC-1, were kindly provided by Prof. Lips (Utrecht, the Netherland). Cells were maintained at 37 °C in 5% CO2 and cultured in T75 flasks filled with 10 mL of F-12K Kaighn’s modification medium (Gibco™ Thermo Fisher Scientific, Waltham, MA, USA). Media was supplemented with 10% heat-activated fetal bovine serum (FBS) (Invitrogen™ Thermo Fisher Scientific, Waltham, MA, USA) and 105 U·L−1 penicillin/streptomycin (EuroClone™, Milan, Italy). Cells were harvested by trypsinization (Trypsin 0.05% and EDTA 0.02%) (Sigma-Aldrich® Merck KGaA, Darmstadt, Germany), resuspended in complete medium, then counted through an optical microscope using a standard haemocytometer before plating. Cells used in all experiments were below 5 passages. All in vitro experiments were monitored for up to 6 days of drug incubation. We performed long-term treatments due to the slow doubling time (about 4 days) of the MTC cell lines.
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