Optimum amount needed is determined from the protocol for lipofectamine 3000. Mdamb transfection kit breast adenocarcinoma altogen. As with every other rnai reagent, sipools require the use of transfection reagents for efficient transfer into the cytoplasm. A comparison showing transfection efficiency of polyjet reagent vs. Introduction lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. L growth medium for a single well of a 24well plate.
Efficient cell transfection with melaminebased gemini. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. Protocol for lipidmediated transient transfection in a549. How to improve the transfection efficiency of a549. While lipofectamine 2000 reagent demonstrated transfection efficiencies only slightly lower those of.
To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. Lipofectamine 2000 to use for a549 transfection researchgate. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells. Assay measure protein expression introduction this protocol shows optimum transfection condition using hilymax in a549 cells.
The a549 cell line is widely used as a model of lung adenocarcinoma, as well as an in vitro model for type ii pulmonary epithelial cells. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Crisprcas9 systems provide a platform for high efficiency genome. Lipofectamine 2000 and 3000 summary in this comparative study, transfections were performed with dnain and compared to the current market leader, lipofectamine 2000 lf2k, using a series of cultured celllines and primary cells. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. A549 chok1 cos7 hek 293 hela hepg2 keratinocytes lncap neuro2a nih 3t3 pc3 fe 3. A sample protocol is listed here for transfection experiments performed in 6well plates. The intracellular trafficking mechanism of lipofectamine. A549 cells are an important model for drug metabolism and are also used as a host for gene transfection. 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.
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. There is a great choice of commercially available transfection reagents, which vary in their efficiency and toxicity in different cell lines. Transfection efficiency in different cancer cells with gfp pdna and mrna by using viromer red. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes.
Viafect transfection reagent transfection assay e4981. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Clathrin and caveolaemediated endocytic pathway contributions to transfection efficiency were studied. However, significant toxicity from lipofectamine 2000 was also reported in u2os cells. Lncap, hepg2, and a549 cell lines in a 96well format, and green fluorescent protein gfp.
Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The i nhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000pgfpn2 transfection. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Effective transfection of cas9 mrna or rnps using lipofectamine 3000 or rnaimax. Invitrogen lipofectamine 3000 transfection reagent fisher. Igur high efficiency transfection on a broad range. The a549 cell line is hypotriploid with a modal chromosome number of 66, which occurs in 24% of cells. The transfection protocol for lipofectamine 3000 reagent was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Transfection efficiency was evaluated with green fluorescence protein gfp expression assays. Hek 293ft, a549 and n2a cells were maintained in dmem medium. Singlecell transfection of adherent cells has been accomplished using singlecell electroporation scep with a pulled capillary. Cas9 rnps delivered into u2os cells using lipofectamine 2000 resulted in up to 80 % modification efficiency at an integrated gfp reporter and cas9 rnps. Improved delivery of cas9 proteingrna complexes using.
Singlecell transfection by electroporation using an. Composites of malonic acid diamides and phospholipids. Choosing the right transfection reagent for optimal efficiency. Characterisation of plasmid dnamrna codelivery in vitro. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Transfection efficiency and protein expression using geneart crispr nuclease vector.
We observed higher efficiency and less toxicity when using itx2. I have been transfecting a549 cells for long time but still could not get transfection efficiency more than 30%. More recently it was shown that proteasomeinhibitors increase peptidebased nonviral gene transfer in certain cell lines. The aggregation behavior of various zwitterionic helper phospholipids, such as dope, dopc, and dppc, in combination with two new cationic lipids, namely th4 and oh4 second generation of malonic ac. Commercially available a549 transfection reagent is provided by altogen biosystems link to manufacturer product page a549 transfection kit. Invitrogen lipofectamine 3000 transfection reagent. American medical association, manual of style, 10th edition, 2007.
We have assessed if proteasome inhibitors doxorubicin and llnl increase lipofectamine 2000mediated gene expression in a549 type ii. Lipofectamine 2000 to use for a549 transfection a volume of 2 ul of lipofectamine 2000 do you think will be enough for an overnight transfection in dmemno pens strep 100 nm sirna. Transient plasmid dna transfection into hela and hacat. Introducing the versatile reagent with the power to transfect your. Transfecting plasmid dna into a549 cells using lipofectamine. Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. Delivery system was used to transfect plasmid dna encoding. The liposome of 1a showed the highest transfection efficiency in a549 cells. To tranfect a549 cells in 24well plate, follow optimum condition for transfection and transfection procedure. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. In contrast, other liposomes 1b and 1c had lower transfection efficiencies as compared with lipofectamine 2000.
Transfection efficiency use our impressive cell database to figure out the viromer which will fit. This reference provides a recommended procedure to transfect plasmid dna into human a549 lung carcinoma cells atcc, cat. I use 24well plates for transfections in triplicates. A549 transfection kit lung carcinoma, ccl185 altogen. Although cas9 rnps can be delivered into mammalian cells via. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
To obtain the maximum transfection efficiency and low nonspecific effects, you. It is possible to plate and transfect the cells on the same day, but cells must adhere to. Under optimized conditions, the c12 and c14 melamine gemini surfactants showed little toxicity and remarkable transfection efficiency, superior to the goldstandard lipofectamine 2000. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. The invitrogen lipofectamine 3000 transfection reagent leverages our. Comparative transfection efficiency of jetprime versus its main competitor. How to maximize transfection efficiency in a549 cells. Transfection protocol lipofectamine 3000 transfection reagent. Cells were treated with pkca aso in the presence of lipofectamine 2000 lipo2000 cationic lipid reagent.
The transfection protocol was optimized appropriately confluence, reagentpcdna ratio, compaction time, and transfection time for each cell line. Selected citations for a549 transfection reagent used in the following publications. Their highest transfection efficiency was only 30% after optimization of the pulse protocols. Improved delivery of cas9 proteingrna complexes using lipofectamine crisprmax. At 72 hours posttransfection the cells were analyzed by western blot for protein expression levels normalized by total protein, 10 g of total protein loaded per each well. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common cells e.
In this chapter, a method of transient transfection of the a549 cell line, human lung cells of alveolar epithelium, with an expression plasmid is described. Each reagent was tested with a range of transfection reagent. Transfection and genome engineering fisher scientific. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results.
Feb 18, 2016 the delivery of purified cas9 protein and grna complexes cas9 rnps has gained increasing attention due to the high cleavage efficiency and potentially lower offtarget effect compared with plasmid dna transfection kim et al. Highest transfection efficiency in many cell types and formats e. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. Transient plasmid dna transfection into hela and hacat cells. Transfecting plasmid dna into a549 cells using lipofectamine 3000 reagent see all transfection protocols. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. We are also pleased to hear trans that trans itx2 will be offered in similar volume configurations to lipofectamine 2000. Rapid and highly efficient mammalian cell engineering via cas9. Lipofectamine 2000 right panel around 5% efficiency. Cyclenbased cationic lipids for highly efficient gene delivery.
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 lipofectamine 3000 transfection reagent. Hepesbuffered physiological saline solution containing pegfp plasmid at a low concentration 0. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Each reagent was used to transfect hek 293, hela, lncap, a549, and. 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. Hela, lncap, a549, and hepg2 cells in 96well format, and gfp expression was analyzed 48 hours posttransfection. Hits were optimized and retested in expanded cell panels. The cell viability and transfection efficiency were. May 15, 2009 they used micropipettes with openings in the 0. Lipofectamine 3000 transfection reagent hepg2 liver.
Reverse transfection is the transfer of genetic material into cells and is reverse because the order of dna and cells is reverse that of conventional transfection. High cotransfection efficiency of plasmid and sirna or mirna reagents. Transfecting plasmid dna into a549 cells using lipofectamine 3000 reagent. Trials of transfection in eukaryotic cells are essential tools for the study of gene and protein function. Dna transfection for luciferase assay in human alevolar. Transfection protocol lipofectamine 3000 transfection reagent a549. 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. For additional information on scaling your transfection reaction, see page 4. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common. Different cell confluencies, dnareagent ratios and total transfection volumes were optimized for two chemical reagents including jetpei and lipofectamine 2000. The vector contained an ofp reporter gene and was transfected with lipofectamine 2000 or lipofectamine 3000 reagent into a u2os and b hepg2 cell lines.
They have been used in a wide range of research fields. Subsequently, the gene transfection of liposome 1a in the presence of fetal bovine serum fbs was investigated fig. L3000001 lipofectamine 3000 transfection reagent sparshbio. Genscript recommends using lipofectamine 2000 for all transfections. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. Transfection protocol lipofectamine 3000 transfection. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. An example of high efficiency forward and reverse transfection of a549 cells using the broad spectrum transitx2 dynamic delivery system can be found in the figure below. It is best to use polypropylene instead of polystyrene tubes. Change the growth medium 4 hours after transfection.