The generation of T-47D ER mutant and WT clones was described in Arnesen et al. (16 (link)). Cells were cultured in RPMI1640 Media (ThermoFisher Scientific) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (ThermoFisher Scientific). Five days prior to all experiments, except those otherwise specified, cells were moved to growth in hormone-deprived media which was composed of phenol red-free RPMI1640 Media (ThermoFisher Scientific) supplemented with 10% charcoal-stripped FBS (ThermoFisher Scientific) and 1% penicillin-streptomycin. Prolonged 25 day E2 treatment of WT cells was performed as described in Arnesen et al. (16 (link)).
Rpmi 1640 media
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China, Canada, Germany, Australia, Japan, Ireland, Belgium, Morocco
RPMI 1640 media is a widely used cell culture medium developed at Roswell Park Memorial Institute. It is a balanced salt solution that provides essential nutrients, growth factors, and other components required for the in vitro cultivation of a variety of cell types, including mammalian, insect, and plant cells.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (586 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (309 mentions)
Streptomycin, by Thermo Fisher Scientific (200 mentions)
Penicillin, by Thermo Fisher Scientific (194 mentions)
L glutamine, by Thermo Fisher Scientific (120 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (87 mentions)
Glutamax, by Thermo Fisher Scientific (68 mentions)
Fetal bovine serum (fbs), by Merck Group (65 mentions)
Dmem media, by Thermo Fisher Scientific (60 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (51 mentions)
Pen strep, by Thermo Fisher Scientific (43 mentions)
Hepes, by Thermo Fisher Scientific (33 mentions)
Penicillin streptomycin, by Merck Group (30 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (30 mentions)
Penicillin, by Merck Group (27 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (27 mentions)
Streptomycin, by Merck Group (26 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (26 mentions)
Lncap, by American Type Culture Collection (26 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (24 mentions)
1 491 protocols using rpmi 1640 media
1
Generation and Culture of ER Mutant T-47D Cells
2
Detailed Protocol for Target Cell Selection
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T2A24 (HLA-A*02:01, HLA-A*24:02, lymphoblast) cells were provided by Dr. Kuzushima (Aichi Cancer Center) [39 (link)]. Jiyoye (HLA-A32, B17, Bw37, Burkitt’s lymphoma), EB-3 (HLA-A3, Aw32, Cw2, Burkitt’s lymphoma), PANC-1 (pancreatic cancer), and ASPC1 (pancreatic cancer) cells were purchased from the American Type Culture Collection (Manassas, VA). T2A24 cells were maintained in RPMI1640 media with HEPES (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biowest, France) and 0.8 mg/ml Geneticin® Selective Antibiotic (Thermo Fisher Scientific, Waltham, MA). Jiyoye and EB-3 cells were maintained in RPMI1640 media (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin (liquid, Thermo Fisher Scientific, Waltham, MA). KP2, KP3, KP4, and SUIT2 cells were purchased from the JCRB Cell Bank (Japan). The expression levels of HLA-A*2402 and mesothelin were examined by flow cytometry with an anti-HLA-A*2402 monoclonal antibody and anti-mesothelin antibody to select HLA-A*2402- and mesothelin-positive target cancer cells.
3
Establishing Tamoxifen-Resistant and Estrogen-Deprived Breast Cancer Cell Lines
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MCF7 cells were sourced from the Michigan Cancer Foundation. Derivatives of MCF7 cells that are resistant to tamoxifen (TamR) or long-term estrogen deprived (LTED) were obtained from Myles Brown (Dana-Farber Cancer Institute) (Bailey et al. 2015) (link). T47D cells were obtained from the American Type Culture Collection. All cell lines have been verified through short tandem repeat profiling, and mycoplasma contamination was excluded. MCF7, TamR and T47D cell lines were cultured in RPMI 1640 media (Thermo Fisher) supplemented with 10% fetal bovine serum (GE Healthcare), 20 mM HEPES (Thermo Fisher) and 0.28 IU/mL insulin (Novo Nordisk). Base media for TamR cells was also supplemented with 5 µM 4-hydroxytamoxifen (Sigma). TamR cells used in experiments were grown in base media without tamoxifen. The LTED cell line was maintained in phenolred free RPMI 1640 media (Thermo Fisher) supplemented with 10% steroid-depleted FBS (Thermo Fisher), 20 mM HEPES (Thermo Fisher) and 0.28 IU/mL insulin (Novo Nordisk). All cells were cultured in a humidified incubator at 37°C with 5% CO 2 and were used at <10 passages post revival. The AR antagonist enzalutamide (Selleck) was re-suspended as a 40 mM stock solution in DMSO and DHT (Sigma) was re-suspended as a 10 µM stock solution in ethanol.
4
Transwell Assay for Cell Migration and Invasion
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Polycarbonate filters (pore size, 8 µm; Corning, Inc.) were used for the Transwell assay. Chamber inserts pre-coated with 200 mg/ml of Matrigel at 37°C for 30 min were used for cell invasion detection, whilst chamber inserts without Matrigel were used for cell migration determination. The upper chamber was plated with 200 µl RPMI-1640 media (Gibco; Thermo Fisher Scientific, Inc.) containing 0.1% FBS and 1×105 A2058 cells treated with different concentrations of PMS (0, 20, 80 and 160 µg/ml) at 37°C for 48 h. The lower chamber was plated with 600 µl RPMI-1640 media (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS. After 48 h incubation at 37°C, cells in the lower chamber were fixed with 4% paraformaldehyde (cat. no. P0099; Beyotime Institute of Biotechnology) at room temperature for 30 min and then stained with 0.5 ml 0.1% crystal violet (cat. no. C0121; Beyotime Institute of Biotechnology) at room temperature for 15 min. migrated or invaded cells were then counted using a light microscope (five fields per chamber; magnification ×200). Each Transwell assay was repeated in five independent experiments.
5
Culturing Prostate Cancer Cell Lines
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Prostate cancer cell lines, DU145 (ATCC® HTB-81™) and LNCaP (ATCC® CRL-1740™), were purchased from American Tissue Culture Collection (ATCC) (Manassas, VA, USA). Both cell lines were cultured in RPMI-1640 media (Thermo Scientific Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 0.1mg/mL streptomycin (Lonza, Walkersville, MD). All cells were propagated in standard cell culture conditions at 37°C, 5% CO2, and 95% humidity. Fresh media was added approximately every 2 days. Upon reaching confluency, cells were detached from flasks using 0.25% trypsin with 0.53 mM EDTA solution, followed by centrifugation to remove trypsin. The cell pellet was then resuspended in fresh media. The Vi-Cell XR cell counting system (Beckman Coulter, Miami, FL, USA) was used to determine the cell number in suspension. The desired cell densities needed for seeding in further experiments were obtained by diluting the cell suspension with fresh media.
6
Casticin Apoptosis Induction Protocol
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Casticin (CTC, Figure 1 A) was purchased from Biopurify Phytochemicals Ltd. (Sichuan, China). Stock solution of CTC (100 mM) was prepared in dimethyl sulfoxide, stored at −80 °C, and diluted in cell culture medium for use. Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), and ribonuclease A from bovine pancreas were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bovine serum albumin was purchased from Biosesang (Sungnam, Korea). RPMI1640 media, fetal bovine serum (FBS), and antibiotic-antimycotic mixture were obtained from Thermo Scientific HyClone (Waltham, MA, USA). ApoScanTM Annexin V FITC apoptosis detection kit was purchased from bio-bud (Seoul, Korea). TUNEL enzyme and TUNEL label were purchased Roche (Basel, Switzerland). BEZ-235 obtained from Selleckchem (Houston, TX, USA). Acryl-bisacrylamide (29:1) was obtained from ELPIS Biotech (Daejeon, Korea).
7
Small Molecule and Peptide Synthesis Protocol
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Chemical reagents for small molecule and peptide synthesis were purchased from various vendors and used as received. The phospholipids were purchased from Avanti Polar Lipids (Alabaster, Al). PBS buffer, DMEM/High glucose media, RPMI 1640 media, and Pen/Strep were purchased from Thermal Scientific (Amarillo, TX). The Gram-positive bacteria (B. subtilis (ATCC 663) and S. aureus (ATCC 6538)) were purchased from Microbiologics (Cloud, MN) as lyophilized cell pellet. E. coli (BL 21) was a gift from the lab of Professor Mary F. Roberts at Boston College. NMR data of the small molecules were collected on a VNMRS 500 MHz NMR spectrometer. MS data were generated by using an Agilent 6230 LC TOF mass spectrometer. Peptide synthesis was carried out on a Tribute peptide synthesizer from Protein Technologies. The fluorescence anisotropy experiments were performed by using a SpectraMax M5 plate reader. Fluorescence images were taken on a Zeiss Axio Observer A1 inverted microscope. Confocal images were taken on the Leica SP5 confocal fluorescence microscope housed in the Biology Department of Boston College. Flow cytometry analyses were carried out on a BD FACSAria cell sorter also housed in the Biology Department of Boston College.
8
Small Molecule and Peptide Synthesis Protocol
9
Lung Cancer Cell Lines Characterization
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The lung cancer cell lines CL1-5 cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY) were established and characterized as previously described [21 (link)]. Lung cancer cell line A549, H23, H1299, and H522 were purchased from the American Type Culture Collection (ATCC). LIJ, a primary cell line from a non-small-cell lung cancer (NSCLC) patient, were kindly provided by Dr. Wu-Chou Su (Institute of Molecular Medicine, National Cheng Kung University, Taiwan). A549 and H1299 cells were culture in Dulbecco’s Modified Eagle Medium (Invitrogen) containing 10% FBS. The other cell lines were all maintained in RPMI-1640 media (Invitrogen) supplemented with 10% fetal bovine serum. All of the cell lines were maintained at 37oC in a humidified atmosphere with 5% CO2. ATCC cell lines were authenticated using short tandem repeat analysis, and all cell lines were tested for free of mycoplasma contamination. WP1130 (Degrasyn) and Vialinin A were purchased from Selleck Chemicals (#S2243) and Tocris Bioscience (#4988), respectively.
10
Cell Line Culturing and Authentication
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Human SNU-245 and SNU-478 cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea) and authenticated prior to receipt. Early passage cells were cultured in RPMI-1640 media (Gibco, Gaithersburg, MD, USA) containing 10% foetal bovine serum (FBS), 10 mM L-glutamine, and Antibiotic-Antimycotic (Thermo Fisher Scientific). Human BTC cell lines HuCCT1, HuH28, and WITT were a gift from Dr. Tushar Patel (Mayo Clinic, Jacksonville, FL), and Mz-ChA-1 was a gift from Dr. Shannon Glaser (Texas A&M Health Sciences Center, Bryan, TX).39 (link) These cells were authenticated through ATCC cell line authentication service (Kit #135-XV). HuCCT1 and HuH28 cells were cultured in RPMI-1640 media (Gibco) containing 10% FBS, 10 mM L-glutamine, and antibiotics. Mz-ChA-1 cells were cultured in CMRL1-media (Gibco) containing 10% FBS, 10 mM L-glutamine, and antibiotics.
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