We optimized conditions for reverse transfection with the HEK 293T cell line. We We have found that Effectene, Lipofectamine 2000 and Fugene 6 work with .Protocol Pub. No. MAN0007824 Rev.1.0 Lipofectamine® 2000 Reagent Protocol Outline A. Plate cells so they will be 70–90% confluent at the time of transfection.In this report we compare luciferase expression and viability in three cell lines that we transfected using four different transfection reagents, the Promega FuGENE® 6 and FuGENE® HD Transfection Reagents, TransIT®-LT1 transfection reagent (Mirus Bio LLC) and Lipofectamine™ 2000 transfection reagent (Life Technologies).
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In a reverse transfection protocol, cells are added directly to a plate MIR2304) and Lipofectamine™ 2000 transfection reagent (Life Technologies.I tried reverse-transfecting HepG2 cells using lipofectamine 2000. I used 750000 It would be great if anyone can share a trusted protocol with me. Thanks.Oct 24, 2017 2) Optimize tranfection reagents( e.g Lipofectamine 2000, Transit 2020 use the reverse transfection protocol and optimize both transfection .
Here, we describe reverse transfection using Lipofectamine® 2000. This protocol is for use with adherent cells, as transfection efficiency for suspension cells .Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol.Both forward and reverse transfection using TransIT-X2® Dynamic Delivery System yield superior luciferase expression compared to Lipofectamine® 2000. Before You Start Akin to forward transfection, optimizing transfection conditions by transfecting a reporter plasmid into the cell type of interest (such as a luciferase or GFP encoding plasmid) into cells is critical prior to high throughout.
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To prepare HEK 293T cells for reverse transfection: 1) 24 hours before needed, plate 10 x 10 6 cells in 10 ml media in a 10 cm dish. Alternatively, 5x10 6 cells can be cultured for 2 days before transfection.Lipofectamine™ 2000 transfection reagent was the most toxic, and produced as high as a 55% drop in viability in HEK293 cells (Figure 2). Jurkat cells appear to be less sensitive to toxicity than HEK293 and Hep G2 cells, as only slight decreases in viability were seen with all transfection reagents in these cells (Figure 4). Figure 4. Reverse transfection of Jurkat cells with pGL4.13[luc2.according to the protocol described (2). Knockdown Experiments, DNA Lipofectamine 2000 and following the reverse-transfection pro- tocol from Invitrogen.
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Jun 23, 2017 This protocol has been used successfully with the cells and conditions outlined below. This reverse-transfection has also been successful Add 0.6 μL Lipofectamine™ 2000 (Invitrogen Cat #11668-019) per 25μL Opti-.Lipofectamine® 2000 Reagent is a proprietary formulation that facilitates highly efficient delivery of Stealth™ RNA molecules or short interfering RNA (siRNA) to mammalian cells for RNAi analysis (1, 2). Here, we describe reverse transfection using Lipofectamine® 2000. Reverse transfection.On the day of transfection, for each well 5 μl of lipofectamine 2000 (Invitrogen) is mixed with 300 μl DMEM without any supplements (Note: No FCS in the medium as this inhibits lipofectamine and siRNA complex formation) and incubated for 10 min at room temperature.