T47D cells were acquired from ATCC and cultured in DMEM (Cellgro) supplemented with 5% FBS and 1% penicillin/streptomycin. PR [24 (link)], STAT5A, and NS [24 (link)] shRNA knockdown cells were created using viral particles (GE/Dharmacon) targeting three different regions of each respective gene. Viral transduction protocol was followed as per manufacturer’s instructions. Transduced, stable cell line pools expressing NS, PR, or STAT5A shRNA were created in T47D cells following 14 days of selection in 2.5 ug/ml Puromycin (MP Biomedicals). Target shRNA sequences are listed in Supplementary Table 1 . Cells were treated with the following reagents where indicated: R5020 (10nM, Sigma) and human prolactin (100ng, Sigma).
R5020
Manufactured by Merck Group
Sourced in United States
R5020 is a laboratory instrument designed for the measurement of progesterone in biological samples. It utilizes a radioimmunoassay (RIA) technique to quantify the concentration of progesterone. The core function of R5020 is to provide accurate and reliable measurements of progesterone levels.
Automatically generated - may contain errors
Lab products found in correlation
Spr4594, by Merck Group (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Puromycin, by MP Biomedicals (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Progesterone p4, by Merck Group (2 mentions)
Mycoalert mycoplasma detection kit, by Lonza (2 mentions)
R5020, by PerkinElmer (2 mentions)
Ru486, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Viral particles, by GE Healthcare (1 mentions)
Human prolactin, by Merck Group (1 mentions)
17β estradiol e2, by Merck Group (1 mentions)
Medroxyprogesterone acetate mpa, by Merck Group (1 mentions)
Estradiol e2, by Merck Group (1 mentions)
4 hydroxytamoxifen 4 oht, by Merck Group (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Penicillin, by Novo Nordisk (1 mentions)
Streptomycin, by Novo Nordisk (1 mentions)
Insulin, by Novo Nordisk (1 mentions)
13 protocols using r5020
1
T47D Cell Line Manipulation and Prolactin Response
2
Breast Cancer Cell Line Maintenance and Hormone Treatments
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The breast cancer cell line T47D was obtained from the University of Colorado Cancer Center Tissue Culture core and was maintained in minimal Eagle’s medium, 5% fetal bovine serum (FBS), 1× non-essential amino acids, 1 × 10−9 M insulin, 0.1 mg/mL sodium pyruvate, and 2 mM L-glutamine. Generation of breast cancer cell lines UCD4 and UCD65 has been previously described [20 (link)]. The UCD4 and UCD65 cell lines were maintained in DMEM/F-12 1:1 with 10% FBS, 1 × 10−9 M cholera toxin, and 1 × 10−9 M insulin. Cell lines were authenticated using short tandem repeat (STR) analysis using the University of Arizona Genetics Core (University of Arizona, Tucson, AZ, USA). All cell lines were routinely tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland). In vitro hormone experiments were performed using phenol red-free media plus charcoal stripped FBS with the same additives described above. Hormone treatment was used as follows: vehicle (0.2% ethanol), 17-β-estradiol (E2), 10−8 M (Sigma-Aldrich, St. Louis, MO, USA); R5020, 10−8 M (PerkinElmer, Waltham, MA, USA); or progesterone (P4), 10−7 M (Sigma-Aldrich), or the combination of E2 plus R5020 (both 10−8 M) for 24 h unless otherwise indicated.
3
Estrogen-Deprived Breast Cancer Cell Lines
4
Cell Line Characterization and Treatments
5
Establishing Stable Cell Lines for Investigating Signaling Pathways
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T47D and BT474 cells were acquired from ATCC and cultured in DMEM (Cellgro) supplemented with 5% FBS and 1% penicillin/streptomycin. PR, STAT1, and STAT2 shRNA knockdown cells were created using viral particles (GE/Dharmacon) targeting three different regions of each respective gene. Viral transduction protocol was followed as per manufacturer’s instructions. Transduced, stable cell line pools expressing NS, PR, STAT1, or STAT2 shRNA were created in T47D cells following 14 d of selection in 2.5 ug/ml Puromycin (MP Biomedicals). Target shRNA sequences are listed in Supplementary Table 1. RFP and STAT1 overexpressing cells were created using lentiviral ORF particles (GE/Dharmacon). Transduced, stable cell line pools overexpressing RFP (control) or STAT1 were created in T47D cell lines stably expressing NS or STAT2 shRNA following 14 d of selection in 15 ug/mL Blasticidin S HCL (Corning). Sequences are shown in Supplementary Table 1. Cells were treated with the following reagents where indicated: R5020 (10 nM, Sigma), human rIFN-α (1000 IU/mL, IFN-α2A, SPR4594; Sigma-Aldrich, vehicle-H20), onapristone (10uM, provided through an MTA with Context Therapeutics), cycloheximide (200ug/mL, Calbiochem), and MG132 (5uM, Selleck Chemical).
6
Breast Cancer Cell Culture and Luciferase Assay
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T47D-WT breast cancer cells carrying one stably integrated copy of the luciferase reporter gene driven by the MMTV promoter (32 (link)) and its derivative clone T47D/A1-2 that also expresses a functional full length rGR (27 (link)) were routinely grown in Dulbecco's Modified Eagle's medium (DMEM, Thermo Fisher Scientific). MCF-7L cells were kindly provided by Carol Lange although originally obtained from C. Kent Osborne (Baylor College of Medicine, Houston, TX, USA) (33 (link)). These cells were maintained in Dulbecco's Modification of Eagle's Medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 1.4 U/ml Insulin (Novo Nordisk® Pharma). For experiments, all cells were plated in DMEM medium without phenol red supplemented with 10% DEXtran-coated charcoal-treated FBS (DCC/FBS) and 48-h later medium was replaced by fresh medium without serum. After 24 h in serum-free conditions, cells were incubated with R5020 (Sigma) 10 nM and/or DEX (Sigma) 10 nM for different lengths of times at 37°C according to each experiment.
7
Calcium phosphate-based HeLa cell transfection
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HeLa cell transfections were carried out by calcium phosphate co-precipitation as described previously [11 (link)]. Briefly, 1.2 × 105 cells were plated in phenol-red free minimum Eagle’s medium (MEM) containing 5% twice charcoal-stripped FBS. Twenty four hours later, cells were washed and treated with the synthetic progestin R5020 (Sigma Chemical Co., St. Louis, MO, USA), the mixed agonist/antagonist RU486, or the pure antiprogestin ZK98299 at the final concentrations indicated in the figures. Cells were harvested in lysis buffer (Promega, Madison, WI, USA), and the lysates were analyzed on a dual luminometer. Results were normalized to Renilla luciferase activity and expressed as indicated in the figures.
8
Breast Cancer Cell Line Cultivation
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Isogenic T47D-A (ER+/PR-A+/PR-B-null) and T47D-B (ER+/PR-B+/PR-A-null) recombinant cells were a generous gift from Dr. Katherine Horowitz (University of Colorado, Denver, CO) and were cultured as described previously All human tumor samples, from female patients, classified as treatment naïve ER+/PR+ primary breast carcinoma were obtained from the Cooperative Human Tissue Network (CHTN). For each tumor tissue selected, the largest dimension was in the range of 1.5 cm–2 cm. Lipofectamine 2000 was from Thermo Scientific (product number 78410). R5020, and RU486 (mifepristone) were purchased from Sigma Aldrich (Saint Louis,MO).
9
High-content screening of phytochemicals
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High-content screening was performed using the Prestwick Phytochemical Library (Prestwick Chemical, Illkirch, France), a collection of 320 pure natural products, mostly derived from plants. Harmol hydrochloride was obtained from Santa Cruz Biotechnology, Inc. Harmine, harmane, R1881, spironolactone, RU486, R5020, dexamethasone, and aldosterone were purchased from Sigma Aldrich (St. Quentin Fallavier, France). Enzalutamide was purchased from Selleckchem (Euromedex, Strasbourg, France), SR12813 from Bio-Techne (Lille, France). All reagents were dissolved in dimethyl sulfoxide (DMSO) at 10 mM before use.
10
Cell Line Characterization and Maintenance
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