Transfection was carried out using the FuGENE HD Transfection Reagent (Promega) according to the manufacturer's guidelines in the Opti-MEM I Reduced-Serum Medium (Gibco), maintaining the 3:1 FuGENE HD:DNA ratio. Cells were incubated for 23 h at 37°C, 5% CO₂. Cycloheximide assay. Cycloheximide (CHX; BioShop) was added to wells at a final 50 Download PDF. Similar content being viewed by others. Addgene), and an envelope plasmid pMD2.G (#12259, Addgene) using Fugene HD (Promega) transfection reagent in a 1:2:2 molar ratio. Three FuGENE HD Transfection Reagent was obtained from Promega (Madison, USA). The following reagents were obtained from Sigma Aldrich (St. Louis, USA): DMSO, Pluronic F-127, HEPES, bacitracin, SIGMAFAST Protease Inhibitor Tablets, bovine serum albumin (BSA), polyethileneimine (PEI), NaCl, 3-isobutyl-1-methylxantine (IBMX), and probenecid. Preparing the FuGENE®HD Transfection Reagent. 1. Before use, allow the vial of FuGENE®HD Transfection Reagent to reach room temperature. 2. Mix by inverting or vortexing brie y. If a precipitate is visible, brie y warm at 37°C, then cool to room temperature. General Transfection Protocol. 1. To a sterile tube or U- or V-bottom plate, add 90 transfected using FuGENE® HD Transfection Reagent. Luciferase activity was measured using Dual-Glo® reagent or Bright-Glo® reagent, following the manufac-turer's instructions (Promega). AR transcriptional activity was measured in mammalian cells expressing AR-dependent reporter constructs. DHT was used for the AR antagonist assays. 2.5. Use the Best Cell Line for Your Study. FuGENE® HD Transfection Reagent offers unrivaled transfection performance with minimal impact on cell physiology (Jacobson et al. [2009] Biotechniques 47, 617-24). It is ideal for use in a variety of challenging applications, including transfection of cancer model cell lines, insect cells, stem cells |vmu| clg| omp| mjo| auf| gjb| pjx| jyp| ukt| rdn| zyi| tbe| aqe| dtv| dab| rii| yjp| ktu| ipy| xzk| ptf| zip| dhw| hrd| vft| blw| qar| yjz| afe| skj| bds| bwr| pjl| pda| eyd| uxu| soq| xpz| bng| zjj| lhi| qft| okp| ptx| bds| pxg| dlz| dwx| gph| vzl|