COS-7 cells were seeded in phenol red-free DMEM (Wisent) supplemented with 5% dextran-coated charcoal stripped serum (Sigma-Aldrich). Twenty-four hours after plating, cells were transfected with plasmid DNA using Fugene HD (Promega) according to the manufacturer’s recommendations. For the transcriptional assays, cells were transfected with 10 ng of pRL-CMV (renilla; internal control, Promega) 25 ng of VP-16-ERα 25 ng of VP-16-ERβ (Addgene38 (link) and 125 ng of 3X ERE-TATA-luciferase (ERE-luc) (Addgene38 (link). Twenty-four hours after transfection, cells were treated with vehicle control and the indicated concentrations of BPS or BPA, as well as 1 nM E2 as positive control. Twenty-four after treatment, cells were lysed using 1X Passive Lysis Buffer (Promega). Luciferase activity was quantified with the Dual Luciferase Assay kit (Promega) using the Glomax96 Luminometer (Promega). Luciferase activity was normalized to Renilla levels and to vehicle control.
Phenol red free dmem
Manufactured by Wisent
Phenol red-free DMEM is a cell culture medium that does not contain the pH indicator phenol red. It is commonly used for applications where the presence of phenol red may interfere with experimental procedures or measurements.
Automatically generated - may contain errors
Lab products found in correlation
Glomax 96 luminometer, by Promega (3 mentions)
Dual luciferase assay kit, by Promega (3 mentions)
Passive lysis buffer (plb), by Promega (3 mentions)
Dextran coated charcoal stripped serum, by Merck Group (2 mentions)
Fugene hd, by Promega (2 mentions)
Paraformaldehyde (pfa), by Polysciences (1 mentions)
Ici 182 780, by Bio-Techne (1 mentions)
Mcf 7, by Wisent (1 mentions)
4 hydroxytamoxifen 4 oht, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Wisent (1 mentions)
Mcf 7 cell, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle s medium, by Wisent (1 mentions)
5 protocols using phenol red free dmem
1
Estrogen Receptor Transcriptional Assay
2
GFP-GR Localization in Cos-7 Cells
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Cos-7 cells were plated on glass coverslips in phenol red free DMEM (Wisent) with 5% charcoal stripped serum (prior to transient transfection with 200 ng pGFP-GR45 (link) using FuGENE 6 [Roche]) as per manufacturer’s instructions. Twenty-four hours post-transfection the cells were treated with the indicated concentrations of DEX and BPA or ethanol control for 6 h. Cells were fixed in 4% paraformaldehyde (Polysciences, Inc.) and mounted on slides with Vectashield 4,6-diamidino-2-phenylindole staining solution (Vector Laboratories). One hundred to two hundred cells were scored per slide in each experiment, by fluorescent microscopy and the data are represented as the average in three independent experiments.
3
Culturing and Treating Cell Lines for Estrogen Assays
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The human hepatocellular carcinoma cell line Huh7 cells was a kind gift from Dr M. Santo and cultured as previously published (Bagu and Santos, 2011 (link)). Human breast cancer MCF-7 cells were purchased from American Type Culture Collection (American Type Culture Collection). MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (Wisent) supplemented with 10% fetal bovine serum (Wisent) and 1% Penicillin-streptomycin antibiotic. Primary human osteoblasts were isolated and cultured as described (Fendri et al., 2013 (link)). For estrogen assays, cells were cultured in phenol red-free DMEM (Wisent) supplemented with 10% charcoal-stripped fetal bovine serum (CS-FBS) for 3 days. The next day, the cells were treated with ethanol (vehicle) or 10−7 M E2 for 24 h. E2 and 4-hydroxy-tamoxifen (4-OHT) were purchased from Sigma-Aldrich, ICI-182, 780 was from Tocris Bioscience. E2, along with its inhibitors ICI-182, 780 and 4-OHT, were reconstituted in 100% ethanol as stock solutions of 2×10−2 M and stored at −20°C as indicated by the manufacturer.
4
PPARγ Transcriptional Assay in COS-7 Cells
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COS-7 cells were seeded in phenol red-free DMEM (Wisent) supplemented with 5% dextran-coated charcoal stripped serum (Sigma-Aldrich). Twenty-four hours after plating, cells were transfected with plasmid DNA using Fugene HD (Promega) according to the manufacturer’s recommendations. For the PPARγ transcriptional assays, cells were transfected with 10 ng of pRL-CMV (renilla; internal control), 25 ng of pcDNA mPPARγ, 25 ng of pCMV6 mRXR, and 125 ng of aP2 enhancer-luciferase (aP2 enhancer-luc). Murine aP2-luciferase was a gift from Bruce Spiegelman (Addgene plasmid #8858). This 520 bp enhancer construct is 5.1 kb upstream from the transcriptional start site and contains a functional PPRE (PPARγ response element). Six hours after transfection, cells were treated with vehicle control and the indicated concentrations of TROG, FM550, TPP, IPTP, DPP, TBB, or TBPH. Twenty-four after treatment, cells were lysed using 1X Passive Lysis Buffer (Promega). Luciferase activity was quantified with the Dual Luciferase Assay kit (Promega) using the Glomax96 Luminometer (Promega). Luciferase activity was normalized to renilla levels and to vehicle control (DMSO).
5
Transcriptional Regulation Assays in 3T3 Cells
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NIH 3T3 or 3T3-L1 cells plated in 12-well cell culture plates in 10% charcoal stripped serum and phenol red free DMEM (Wisent). Twenty-four hours later the cells were transfected with Fugene 6 or Fugene HD (Roche Diagnostics), respectively, according to the manufacturer’s instructions. For GRE-Luc and MMTV-Luc, previously described, the cells were transfected with 125 ng of the reporter plasmid, 25 ng pTL2-GR, and 10 ng pRL-CMV as an internal control. For C/EBPα promoter activity the cells were transfected with 125 ng of the C/EBPα-luciferase reporter gene (−350/+7), 25 ng pTL2-GR, and 25 ng of C/EBPβ cDNA. aP2-Luc activity was assessed using 125 ng of aP2-Luc with 25 ng of pMSV-C/EBPβ cDNA or pMSV-C/EBPδ and 12.5 ng pTL2-GR. In all cases DNA concentrations were brought up to 500 ng/well using sheared salmon sperm DNA (Life Technologies). Following transfection, cells were treated with the indicated concentrations of DEX, BPA or ethanol control for 16–24 h after which the cells were lysed in Passive Lysis Buffer (Promega) and the luciferase activity was assessed using the Dual Luciferase Assay kit (Promega) in a Glomax96 luminometer (Promega).
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