Caco 2 transfection lipofectamine 2000 manual

Gfp expression was assayed by fluorescence microscopy in hep g2 cells 24 h after transfection. Transfection protocol lipofectamine 3000 transfection reagent calu6. Luciferase expression was compared at 24 hours post transfection using a standard luciferase assay. We would recommend using our fitcconjugated control sirna sc36869 to measure transfection efficiency of. Small antisense oligonucleotides against gquadruplexes. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. There are no other equine cell lines available unfortunately, so i have to use this one. Renilla luciferase vector prltk, promega was cotransfected with each plasmid construct as an internal control. Transfection protocol of adherent caco2 cells 96well plate. Co transfection of two plasmids need the protocol for lipofectamine 2000 based transfection nov 012009 hi, i am trying to cotransfect transient trasnfection two different plasmids in to hek293t cells. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. The peak luciferase expression was observed at 9 days post transfection, but the expression sustained over three weeks. Plate cells so they will be 7090% confluent at the time of transfection.

Lipofectamine 2000 transfection reagent from invitrogen. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. Lipofectamine 2000 reagents 3t3 4 4t1 2 a431 2 a549 3 achn 2 bend. The peak luciferase expression was observed at 9 days post transfection. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Caco2 cells were cultured in 100mm petri dishes sarstedt in dmem. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. For some cell types, such as cos7, the transfection efficiency was over 95% while other hardertotransfect cells, such as caco 2, displayed efficiencies around 75%. Caco 2 cells were seeded 21 days for differentiation before transfection in sixwell plates. The hrfc and hpcft promoterluciferase reporter constructs were transfected into caco 2 cells grown in 12well plates at less than 50% confluence with 2.

How does transfection with lipofectamine 2000 work. The protocol works efficiently and reproducibly for the transfection of dna and protein in mdck, arpe19 and caco2 cells, preserves all. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Differentiated caco 2 cells were transfected with different sirnas with silencemag sirna delivery reagent cat. Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco2 colon cancer cell line. Lipofectamine 2000 reagent thermo fisher scientific. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Dna constructs of mlck promoters were transiently transfected into caco 2 cells using transfection reagent lipofectamine 2000 life technologies. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in a 96well format, and. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Caco2 cells passage 2538 were used for all experiments. Compare caco2 cell line transfection from leading suppliers on biocompare. Transfecting plasmid dna into caco2 cells using lipofectamine.

Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. To transfect cells with sirna, follow the protocol as. Dosedependent targeted suppression of pglycoprotein. Highest transfection efficiency in many cell types and formats e. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. A polymerbased transfection caco2 kit for colon adenocarcinoma cells yields at least 94% transfection efficiency and is a helpful tool for gene expression studies as well as for assessing cellular functionality. Gfp, using lipofectamine 2000 life technologies or k2 transfection system biontex, munich, germany following manufacturer instructions. I have tried transfecting the cells using lipofectamine 2000 and 3000 but the transfection efficiency. Does anyone have protocols to transfect caco2 using fugene. We have not specifically evaluated the transfection efficiency of this transfection reagent with the caco 2 cell line. Lipofectamine 2000 reagent protocol 20 2 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000.

Has anyone tried atccs transfex transfection reagent. I have tried transfecting the cells using lipofectamine 2000 and 3000 but the transfection efficiency is very low transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Invitrogen lipofectamine 2000 transfection reagent 15ml. Transfection is the process of introducing foreign dna into the cells either by. I was recently tasked with developing a crispr protocol for primary and bonederived. The lenti293t cell line has been tested as a suitable cell line for lentiviral production due to high transfection efficiency, high level of protein yield and high titer of lentiviral production.

Lipopolysaccharide induced increase in caco2 intestinal. The growth medium was dulbeccos modified eagle medium dmem containing 1. Max % of positive cells after transfection of a gfpplasmid 3. On the day of transfection just prior to preparing complexes, plate 1. For each transfection sample, prepare dna lipofectamine 2000 complexes as follows. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Lipofectamine 2000 transfection reagent dartmouth college. Genetic transfection an overview sciencedirect topics. Differentiationdependent regulation of the intestinal. Invitrogen lipofectamine 3000 transfection reagent 1. This reference provides a recommended procedure to transfect plasmid dna into caco 2 human colorectal adenocarcinoma cells atcc cat. Dynamic delivery system mirus bio and lipofectamine 2000 transfection. Caco2 human colon adenocarcinoma cells were seeded from 90100% confluent culture the day before transfection with the density 10,000 cellswell in 100. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection.

It has also been optimized to work for many mammalian cell lines. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Do you have any optimized protocol for this purpose. The main advantage of using lipofectamine 2000 however, was its ability to transfect a range of very different cell types coupled with a high efficiency of transfection. Lipofectamine 2000 transfection reagent from thermo fisher. Bmal1 controls glucose uptake through pairedhomeodomain.

Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Transfection protocol of adherent caco2 cells 96well plate 1. However, at div2, it became difficult to have good transfection. Human intestinal caco 2 cells were transiently transfected with cdx2 mammalian expression vector using amaxa electroporation. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines.

Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Cotransfection of two plasmids tissue and cell culture. Lipofectamine 2000 from invitrogen biocompare product. Efficient electroporation of dna and protein into confluent and. To transfect cells with sirna, follow the protocol as described for dna but.

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