CV-1 cells were seeded into 24-well plates and cultured for 24 h before transfection. Prior to transfection, the medium was replaced with 10% charcoal dextran-treated FBS–DMEM. After 4 h, a DNA mixture containing an MMTV-luciferase reporter plasmid (pGL4.36[luc2P/MMTV/Hygro] vector, 0.3 μg, Promega) with (TGTTCT)6 as the enhancer element sequence, and an internal control plasmid pRL-SV-40 (5 ng), was transfected using the TransFast reagent (Promega, Madison, WI, USA). After transfection, cells were treated with 10 μg/mL compounds (samples) or 1 μM spironolactone. After 2 h, the treated cells were further treated with 1 nM dexamethasone and incubated for an additional 24 h. The luciferase activities of the cell lysates were measured using the Dual-Luciferase Reporter Assay System, according to the manufacturer’s instructions (Promega). The relative luciferase activity was normalized to the corresponding Renilla luciferase activity to determine transfection efficiency.
Transfast reagent
Manufactured by Promega
Sourced in United States
TransFast is a transfection reagent that facilitates the delivery of nucleic acids, such as plasmids and RNA, into eukaryotic cells. It is designed to enable efficient and reliable transfection of a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (5 mentions)
Lsrii flow cytometer, by BD (3 mentions)
X tremegene hp dna transfection reagent, by Roche (3 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Pgl3 basic plasmid, by Promega (2 mentions)
Prl tk, by Promega (2 mentions)
Dual glo luciferase kit, by Promega (2 mentions)
Prl tk plasmid, by Promega (2 mentions)
Pgl4.36 luc2p mmtv hygro vector, by Promega (1 mentions)
Multimode microplate reader, by Berthold Technologies (1 mentions)
One glo luciferase assay system, by Promega (1 mentions)
Pgl3 promoter, by Promega (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter gene assay system, by Promega (1 mentions)
Pcbascei, by Addgene (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
31 protocols using transfast reagent
1
Luciferase Assay for MMTV Promoter
2
Quantifying HIF Activity in AgNP-Treated MCF7 Cells
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MCF7 cells (grown in a 10 cm tissue culture dish) were transfected with 5 μg of HRE-dependent luciferase reporter construct pGL4.42 or a control construct pGL3-promoter (Promega Corporation, Madison, USA) using the Trans-Fast™ Reagent (Promega Corporation) according to the manufacturer’s instructions (pGL4.42 and pGL3 vector maps are provided in Figure S1 ). After 24 hours, the transfected cells were disassociated and plated in 96-well plates (5×103 cells/well). The cells were incubated for 3 hours to be allowed to adhere before being treated with the indicated concentrations of AgNPs (0, 20, 40, 60, 80, and 100 μg/mL in medium). HIF activity was induced by incubating the cells in hypoxic (0.1% O2) conditions or by treating the cells with cobalt chloride (CoCl2, 250 μM) for 16 hours. The luciferase activity was measured after 2 minutes by adding 100 μL of ONE-Glo™ luciferase assay system (Promega Corporation) according to the manufacturer’s instructions using a Multimode Microplate Reader (Berthold Technologies, Bad Wildbad, Germany).
3
Evaluating SPINK5 Promoter Activity
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CV-1 cells were inoculated into 24-well plates and cultured for 24 h before transfection. Cells were cotransfected with SPINK5pro-Luc reporter plasmid (constructed in a previous study [28 (link)]) and a control plasmid pRL-SV-40 using TransFast reagent (Promega, Madison, WI, USA) and cultured for 24 h. Then, the cells were treated with LCE or LCE components at the indicated concentrations and lysed after 24 h using a passive lysis buffer (Promega). Luciferase assays were performed with the Dual-Luciferase® Reporter Assay System (Promega), according to the manufacturer’s instructions. Relative luciferase activities were calculated with respect to Renilla luciferase activities.
4
TGFβ and Triapine Modulate NHEJ
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SKOV-3-DR-GFP cells were pretreated with 5 ng/mL or 20 ng/mL TGFβ for 24 h before being transiently transfected with the empty vector pcDNA/Neo (Thermo Fisher) or the I-SceI endonuclease expression vector pCBASceI using the TransFast reagent (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Five hours after transfection, cells were retreated with 5 ng/mL TGFβ, 20 ng/mL TGFβ, or 0.75μM triapine for 48 h. Cells were trypsinized and analyzed for the percentage of GFP-positive cells by flow cytometry using a LSR II flow cytometer and FlowJo software (Version 10.4; BD Biosciences, East Rutherford, NJ, USA).
5
Specificity Assessment of Anti-PP5 Antibody
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RNAi approach was used to test the specificity of the anti-PP5 polyclonal antibody. dsRNAs targeting the bacterial kanamycin gene (negative control) or the PpD3 gene encoding PP5 (either the coding sequence (CDS) or 3′-UTR) were designed. dsRNAs were synthetized using the MEGAscript T7 transcription kit (cat # AM1334, Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer. dsRNA-treatment was performed, as described earlier [32 (link)]. Briefly, D.Mel-2 cells were cultured in 6-well plates and transfected with 10 μg dsRNA using TransFast reagent (cat # E2431, Promega, Madison, WI, USA) for 3 days at 25 °C. Oligonucleotide primers used in this study are shown in Table S1 .
6
Differentiation and Transfection of 661W Cells
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661W cells (mouse retinal progenitor cells) were cultured in DMEM (Cellgro, Mediatech, Inc., Manassas, VA) supplemented with 10% FBS (Atlanta Biologicals, Norcross, GA) and 1% Antibiotic/Antimycotic (Cellgro, Mediatech, Inc.) at 37 °C and 5% CO2. Treatment with 316 nM staurosporine for 24 h induced 661W differentiation [124 (link)]. For transfection, prewarmed media containing 2 µg each of the GFP-BAX, mitoBFP, and HDAC3-Flag plasmids was mixed with 2 volumes of Transfast reagent (E2431, Promega, Madison, WI), vortexed and incubated at room temperature for 15 min. After removal of regular media, the transfection mixture was added to cells and they were incubated at 37˚C for 1 h, after which fresh media was added to the cells and they were incubated for a further 15-24 h. Overexpression of HDAC3-Flag is selectively toxic to differentiated neurons [125 (link)], including differentiated 661W cells [31 (link)] and plays a critical role in the apoptotic program executed by RGCs [57 (link), 59 (link)]. 661W cells, originally described as RGC-5 cells from Rattus norvegicus, were confirmed in our laboratory as Mus musculus origin by PCR of the mitochondria d-loop [126 (link)] and the upregulation of TUBB3 after differentiation [31 (link)].
7
DNA Repair Mechanism Study in SKOV-3 Cells
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SKOV-3-DR-GFP cells were established as described previously (Lin et al, 2014 (link)); cells were transfected with the empty vector pcDNA-Neo or the I-SceI endonuclease expression vector pCBASceI (provided by Dr Maria Jasin, Memorial Sloan-Kettering Cancer Center) (Pierce et al, 1999 (link)) using the TransFast reagent (Promega, Madison, WI, USA) according to the manufacturer's protocol. Five hours after transfection, cells were treated with various concentrations of triapine for 48 h. For siRNA transfection, cells were transfected with 50 nM siRNA using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) for 16 h before transfection with pcDNA-Neo or pCBASceI plasmid and incubated for 48 h. Thereafter, cells were trypsinised and green fluorescence and side scatter of 50 000 cells were analysed by flow cytometry using an LSR II flow cytometer (BD Biosciences; San Jose, CA, USA). The cell population with an increase in GFP above the baseline level was gated to determine the percentage of GFP-positive cells. Data analyses were performed using the FlowJo software (Tree Star, Ashland, OR, USA). BRCA1, Rad51, and R2-RNR siRNAs have been described previously (Lin et al, 2004 (link), 2014 (link)). BRCA2-siRNA (SMARTpool, siGENOME) was purchased from GE-Dharmacon (Lafayette, CO, USA).
8
Nrf2 Activation by 7-ML in HepG2 Cells
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An antioxidant response element (ARE) promoter fragment (697 bp) of human the NQO1 promoter was extracted from the genomic DNA of HCT116 human colorectal cancer cells and inserted into the pGL3-Basic plasmid (Promega, Madison, WI, USA). HepG2 cells were seeded in a 24-well plate at a density of 6 × 104, incubated for 24 h, and transiently cotransfected with the ARE-Luc reporter plasmid or the control plasmid pRL-SV40 using the TransFast™ reagent (Promega). Transfected cells were treated with 7-ML for 20 h and then with tBHP (tert-butyl hydroperoxide; 200 μM) for 2 h. Firefly and Renilla luciferase activities were then determined using a Dual-Luciferase reporter gene assay system (Promega). Nrf2 (nuclear factor-erythroid 2-related factor 2) luminescence signals were normalized versus SV40 (Renilla) luciferase activity. As a positive control for ARE-luciferase activity, sulforaphane was used [33 (link)].
9
Assessing DNA Double-Strand Break Repair
10
Exploring the Impact of Six1 on Human Keratinocytes
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