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. Viafect transfection reagent transfection assay e4981. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. This result shows that simple, lipidmediated transfection of hep g2 cells is a viable alternative to gene. Low levels of gsta1 expression are required for caco2. Fugener hd transfection reagent technical manual tm328. Optimization of lama1 gene amplification and transfection. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Transfex transfection of plasmid dna into caco2 cells. Lipofectamine 2000 from invitrogen biocompare product. The reagent provides high transfection efficiency in many cells types and formats. Transitx2 dynamic delivery system mirus bio and lipofectamine 2000 transfection reagent thermo fisher scientific were used to transfect plasmid dna encoding luciferase into 41 different cell types at three reagenttodna ratios. To transfect cells with sirna, follow the protocol as described for dna but.
The transfection protocol for lipofectamine 3000 reagent was developed to be easy to use while still. A b on the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using invitrogen lipofectamine 3000 transfection reagent. C caco2 cells were transfected with gfpkras4b and sirna control or targeting gpr31. Cellspecific transfection protocols thermo fisher scientific uk. General considerations for using the transfex transfection reagent. Incubate transfection complexes at rt for 15 30 minutes 5. 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. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Transitx2 dynamic delivery system and lipofectamine 2000 transfection reagent were used to transfect plasmid dna encoding luciferase into 30 different cell types at three reagenttodna ratios. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. Follow these important guidelines when transfecting dna into caco2 cells using lipofectamine ltx reagent. A sample protocol is listed here for transfection experiments performed in 6well plates.
The efflux transporter pglycoprotein pgp, encoded by the abcb1 gene, decreases the bioavailability of a wide range of orally administered drugs. Transfection of human intestinal caco2 cells with metafectene easy hannah schneider internal medicine iv, im neuenheimer feld 345, university of heidelberg introduction the human epithelial cell line caco2 is derived from a colorectal adenocarcinoma and serves. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Caco 2 cells were seeded 21 days for differentiation before transfection in sixwell plates. This protocol is for transfecting plasmid dna into mammalian cells. Iian advanced protocol for transfecting caco2 cells 100 l 500 l l part ii. Transfection is the process of introducing foreign dna into the cells either by physical. Magnetofectamine o2 transfection kit oz biosciences.
Superior performance for co transfection of sirna and plasmid dna. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to deliver invitrogen. The caco2 cell line in particular is a wellestablished model. Bmal1 controls glucose uptake through pairedhomeodomain. Invitrogen lipofectamine transfection reagent protocols have been. This reagent reduces the size of transfection complex, therefore increasing transfection efficiency. Culture vessel multiplication factor shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection.
Caco2, human colorectal adenocarcinoma, plasmid dna, lipofectamine 3000. T84 cells have been successfully transfected with an antisense regulator of cftr. Plasmid dna and sirna transfection of intestinal epithelial. If you wish to use antibiotics during transfection, test your conditions thoroughly. Jun 03, 20 the efflux transporter pglycoprotein pgp, encoded by the abcb1 gene, decreases the bioavailability of a wide range of orally administered drugs. Dna is enclosed within the liposomes and the complexes fuse with the host cell membrane for dna entry into the cytoplasm and eventu ally into the nucleus. This kit replaces and outperforms the original magnetofectamine which was the combination of lipofectamine 2000 from life technologies corporation and combimag transfection reagent. International journal of pharmaceutics 315 2006 1223 123 troporation has become a common laboratory tool for in vitro transfection of bacterial, plant, and mammalian cells with plasmid dna neumann et al. Transfection protocol of adherent caco2 cells 96well plate. Drug permeability studies using the in vitro caco 2 cell model commonly rely on small molecule modulators to. Has anyone optimised the transfection of caco2 cells using. Comparison of pdna transfection efficiency in human colon carcinoma caco2 cells transfected with viromer one red and a liposomebased reagent metafectene. Caco2 transfection reagent colon carcinoma cells altogen.
Has anyone optimised the transfection of caco 2 cells using the neon transfection kit. Maintain p19 cells undifferentiated in mem with 10% serum 7. The aim of the research was to optimise the amplification and transfection conditions of lama1 gene. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. For transfections, caco 2 cells 10 6 cells per 6 cm 2 dish were transiently transfected in suspension using lipofectamine 2000 as recommended by the manufacturer. Lipopolysaccharide induced increase in caco2 intestinal. C 0 genjet in vitro dna transfection reagent for caco2 cells. Lipofectamine 2000 transfection reagent dartmouth college. Transfex transfection of plasmid dna into caco2 cells caco2 atcc cat. To determine the optimal transfection conditions, 5 ng of each sirna 20 m stock and sirna control were transfected using the following ratios wtvol of sirna to transfection reagent. The shortchain fatty acid butyrate was readily taken up by caco2 cells. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
The cell membranes of animal cells are composed of a bilayer of phospholipids with hydrophilic surfaces facing the cytoplasm and extracellular environment figure 11. A highly efficient method of transfecting hep g2 cells. Validation of antibody specificity using reverse short. To transfect cells with sirna, follow the protocol as.
Efficient transfection of dna or shrna vectors into neurons. Drug permeability studies using the in vitro caco2 cell model commonly rely on small molecule modulators to estimate the. A successful transfection of caco2 cells with nanoparticles was observed. Lipofectamine ltx dna transfection reagent protocol. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. The advanced protocol for hardtotransfect cells is provided only if general protocol gives less than 10%.
Nov 29, 2007 efficient and longlasting transfection of primary neurons is an essential tool for addressing many questions in current neuroscience using functional gene analysis. This reference provides a recommended procedure to transfect plasmid dna into caco2 human colorectal adenocarcinoma cells atcc cat. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. The proprietary formulation of lipids and polymers ensures the maximum possible transfection efficiency and cell viability. Genjet in vitro dna transfection reagent for caco2 cells ver. Improving transient transfection efficiency in a differentiated, polar. See page 2 to view a typical plasmid transfection procedure. Transient transfection of epithelial monolayers provides a powerful system in which to express the cdna of any epitheliumspecific protein transiently in a native polarized. Transitx2 dynamic delivery system and lipofectamine 2000 transfection reagent were used to transfect sirna targeting endogenous proteins gapdh and aha1 or to deliver a nontargeting control in primary normal human dermal fibroblasts nhdf. Transfection enhancer reagent 1224 hours after transfection if the viability of caco2 cells being transfected is affected at 16 24 hours post transfection, the level of cytotoxicity can be decreased by changing the growth medium and. The followinglls were daincubatey ced in medium without antibiotics while the transfection mix was prepared. Lipofectamine 2000 lf2000 and jetpei pei were supplied by invitrogen and polyplustransfection, respectively. Dosedependent targeted suppression of pglycoprotein.
Invitrogen, and plasmid transfection with lipofectamine 2000 or. A polymerbased transfection caco 2 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. Lipofectamine 2000 transfection protocol 10cm plates reagents. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically lipofectamine plus and lipofectamine 2000.
Preconfluent cells were transfected with nonspecific negative control sirna ns sirna or gsta1specific sirna gst sirna. Highest transfection efficiency in many cell types and formats e. Compare caco2 cell line transfection from leading suppliers on biocompare. Caco2 cells human colon adenocarcinoma were obtained from the european collection of animal cell cultures ecacc. Lipids are a broad class of fatsoluble biomolecules, such as fats, oils, and waxes. Polyplustransfections strengths are 20 years of experience in manufacturing transfection solutions with tailored scientific and regulatory support to accelerate research. Gfp, using lipofectamine 2000 life technologies or k2 transfection system biontex, munich, germany following manufacturer instructions. This assay is commonly used to study and downregulate the expression of gene of interest 6. Transfection by cationic gemini lipids and surfactants.
Highest transfection efficiency in cells at 90% confluency. Transitx2 dynamic delivery system was simple to use, 23 times better for. Other transfection reagents, including effectene, fugene, as well as. The alliance of mtx transfection reagent and combimag leads to increased transfection efficiency, minimized toxicity and enhanced gene expression. Invitrogen lipofectamine 2000 transfection reagent 15ml. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Transient transfection of polarized epithelial monolayers with cftr. Compare caco 2 cell line transfection from leading suppliers on biocompare. Itx2 dynamic delivery system mirus bio and lipofectamine 2000 transfection reagent thermo fisher scientific were used to transfect sirna targeting endogenous proteins gapdh and aha1 or to deliver a nontargeting. I have tried transfecting the cells using lipofectamine 2000 and 3000 but the. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Plate cells so they will be 7090% confluent at the time of transfection. A successful transfection of caco 2 cells with nanoparticles was observed. The addition of antibiotics to media during transfection may result in cell death, and has not been tested for caco2 cells. Amaxa, neon, lipofectamine ltx, magnetic nanoparticle transfection. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Atcc achieved transfection efficiencies of 70%, using the protocol described below.
Unless otherwise stated all other materials were sourced from sigma. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofection, also known as lipid transfection or liposomebased transfection, uses a lipid complex to deliver dna to cells. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology.
Genetic transfection an overview sciencedirect topics. Transfection protocol lipofectamine 3000 transfection. Add the dna cocktail transfection buffer mix dropwise to the cells some protocols recommend waiting 1530mins for precipitate to form but i get better transfection when i dont wait 5. Transfection protocol of adherent caco2 cells 96well plate 1. Each tube gets 8mls of optimem, 32ug of gag pol, 12. For additional information on scaling your transfection reaction, see page 4. 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. Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco2 colon cancer cell line. Transfection of caco 2 cells with antimir429 against mir429 blocked the lps induced. Before seeding cells for virus package, make sure hek293t cells are in rapid replication state by growth cells to approximately 75%80% confluence in a 10 cm cell culture dish and passaging at 1. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. For some cell types, such as cos7, the transfection efficiency was over 95% while other hardertotransfect cells, such as caco2, displayed efficiencies around 75%. Dynamic delivery system achieves higher knockdown than lipofectamine 2000.
Dynamic delivery system mirus bio and lipofectamine 2000 transfection. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Lipofectamine 2000 and lipofectamine rnaimax transfection. I use 24well plates for transfections in triplicates. Htb37 is an epithelial, human colorectal adenocarcinoma cell line. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Transfection of human intestinal caco2 cells with metafectene. Caco2 cell avalanche transfection reagent human colon. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Mammalian cells transfection protocoljavier calvo follow invitrogen lipofectamine 2000 protocol. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Higher densities can lead to spontaneous differentiation.
Transient transfection of polarized epithelial monolayers. Sep 01, 2018 lipidoids have also been used with dna, for example 14n87 92d, which was more active than the standard lipofectamine 2000 in the transfection of hela cells with. Advanced protocol for transfecting hardtotransfect mammalian cells important. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. I have tried transfecting the cells using lipofectamine 2000 and 3000 but the transfection efficiency is very low transfection method.
Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Does anyone have protocols to transfect caco2 using fugenelipofectamine. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to study cellular functions of proteins of interest, studies to identify nontoxic gene delivery reagents are limited. Genscript recommends using lipofectamine 2000 for all transfections. The products included in this piece are our most robust transfection reagents that provide solutions to both common and hardtotransfect cell types, promoting effective. Based on the science park close to strasbourg france, polyplustransfection offers an extensive and growing range of transfection reagents available worldwide. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Evaluation of transfection methods for transient gene. Does anyone have protocols to transfect caco2 using fugene. Cellspecific transfection protocols thermo fisher scientific us.
Transfection on caco 2 cells with lipofectamine 2000 revealed increasing fluorescence intensity with increasing volumes while slight relative increase in fluorescence intensity compared to the control was detected using 1 mgml polyethylenimine. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. In this thesis, we used a different technique, short interfering ribonucleic acid sirna transfection assay, to analyse the antibody specificity. Caco2 and thp1 should be simple to transfect if you have the appropriate reagents tailored. Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco 2 colon cancer cell line.
Prepare plasmid dnalipid complexes recommend 2 doses of lipid. In the case of confluent caco2 cell layers, the use of lipofectamine 3000 transfection reagent lanes b1b2 and c1c2 in fig. Lipofectamine 2000 reagent thermo fisher scientific. Mechanistic studies on the uptake and intracellular. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Caco2 cell avalanche transfection reagent is a new class of unique chemical formulations specifically formulated and optimized for transfecting caco2 cells. Caco2 2 caki1 4 chok1 1 chos 1 colo 205 4 cos7 4 du 145 2 h460 3 h9c2 3 hcc1937 5 hct116 1. Lipofectamine 2000 are the most efficacious and showed little or no.
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