Improved delivery of cas9 proteingrna complexes using. The day before transfection, dissociate cells that are 8090% confluent in a t75 flask. Transfection of a549 cells use this procedure to transfect plasmid dna into a549 cells in a 24well format for other formats, see scaling up or down transfections, below. Change the growth medium 4 hours after transfection.
Lipofectamine 2000mediated gene expression in a549 type ii pneumocytelike and cfte cells cystic fibrosis tracheal epithelium. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Lipofectamine 2000 reagent thermo fisher scientific. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. We observed higher efficiency and less toxicity when using itx2.
Cells were grown in 96well plates and transfected 0. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Arpe19 transfection kit retinal pigment epithelium. All amounts and volumes are given on a per well basis. How to maximize transfection efficiency in a549 cells. Genscript recommends using lipofectamine 2000 for all transfections. Introduction lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. When we compare the scepbased transfection to the control experiment of bulk transfection mediated by lipofectamine 2000, we notice an average brighter egfp fluorescence for the bulk method than the scep method.
It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. L3000001 lipofectamine 3000 transfection reagent sparshbio. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common cells e. Add 6l of lipofectamine2000 from biostores and 100 l of. Comparative transfection efficiency of jetprime versus its main competitor.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. A549 transfection kit lung carcinoma, ccl185 altogen. Transfection protocol of adherent a549 cells 96well plate. I have been transfecting a549 cells for long time but still could not get transfection efficiency more than 30%. Invitrogen lipofectamine 3000 transfection reagent.
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. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. The day before transfection, trypsinize and count the cells. Each cell line was transfected with a luciferase reporter plasmid using either viafect transfection reagent, lipofectamine 2000 transfection reagent or lipofectamine 3000 transfection reagent following manufacturer instructions, applying either 5l 50ng of dna or 10l 100ng of dna of the transfection complex in each well of a 96. The intracellular trafficking mechanism of lipofectamine. Trials of transfection in eukaryotic cells are essential tools for the study of gene and protein function. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. The protocol indicated below is a reverse transfection protocol. Transfecting plasmid dna into a549 cells using lipofectamine. Transfection and genome engineering fisher scientific. Lipofectamine 3000 transfection reagent hepg2 liver. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Singlecell transfection by electroporation using an. Introducing the versatile reagent with the power to transfect.
This reference provides a recommended procedure to transfect plasmid dna into human a549 lung carcinoma cells atcc, cat. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. Lipofectamine crisprmax is more robust than lipofectamine rnaimax and several other transfection reagents in delivery of cas9 proteingrna complexes into a variety. Prewarm 50ml of optimum stored in cold room at 4c 10min. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Invitrogen lipofectamine 2000 transfection reagent. They have been used in a wide range of research fields.
Each reagent was used to transfect hek 293, hela, lncap, a549, and. Lncap, hepg2, and a549 cell lines in a 96well format, and green fluorescent protein gfp. To tranfect a549 cells in 24well plate, follow optimum condition for transfection and transfection procedure. Original article effect of mir16 on proliferation and. Spb 1kb promoter region and ttf1 wildtype wt plasmid were cotransfected in a549 cells using either biontex k2 or lipofectamine 2000 transfection. A sample protocol is listed here for transfection experiments performed in 6well plates. Enhanced transfection efficiency using lipofectamine 3000 reagent.
Transient plasmid dna transfection into hela and hacat. Plate cells for transfection only 1 day before the experiment. We are also pleased to hear trans that trans itx2 will be offered in similar volume configurations to lipofectamine 2000. For additional information on scaling your transfection reaction, see page 4. Transfection efficiency was determined by realtime rtpcr. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000. Assay measure protein expression introduction this protocol shows optimum transfection condition using hilymax in a549 cells. Viafect transfection reagent transfection assay e4981. Reverse transfection is the protocol of choice for most applications, as it frequently yields better transfection efficiency and facilitates the experimental protocol by combining cell seeding and. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. 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.
Commercially available a549 transfection reagent is provided by altogen. Transfection protocol lipofectamine 3000 transfection. A comparison showing transfection efficiency of polyjet reagent vs. Transfecting plasmid dna into a549 cells using lipofectamine 3000 reagent. I have used lipofectamine and fugene for plasmid transfection. Gibco rpmi 1640 with glutamax supplement 61870036 10% gibco fbs a3160401 1. 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. Lipofectamine 2000 dna transfection reagent protocol. One can get over 4050% transfection efficiency with plasmid dna and over 85% with sirna. 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. Feb 18, 2016 lipofectamine crisprmax is more robust than lipofectamine rnaimax and several other transfection reagents in delivery of cas9 proteingrna complexes into a variety of cell lines. In biontex experiments gfp positivity increased to. To perform transfection experiments in other cell culture plates. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate.
N v i s i on io 20 transit certified transfection reagents. Transfection protocol lipofectamine 3000 transfection reagent a549 lung cancer cells complete growth medium component cat. Jun 15, 2016 trials of transfection in eukaryotic cells are essential tools for the study of gene and protein function. 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. Dna transfection for luciferase assay in human alevolar. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. When we compare the scepbased transfection to the control experiment of bulk transfection mediated. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Optimized conditions for successful transfection of human. 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. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent.
Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Higher densities can lead to spontaneous differentiation. 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. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Transfection protocol transfection components component cat. Optimization procedure for small interfering rna transfection in a 384well format. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well.
Transfection protocol of lipofectamine rnaimax can be. Reverse transfection is the protocol of choice for most applications, as it frequently yields better transfection efficiency and facilitates the experimental protocol by combining cell seeding and transfection into a single step. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. A549 lipofectamine 3000 lipofectamine 2000 fugene hd lipofectamine 2000 lipofectamine 3000 competitor 1 competitor 2 western blot, hepg2 gststat. May 15, 2009 comparison with lipofectamine 2000 mediated bulk transfection. However, delivery of the cas9 rnps by neon electroporation was effective across all different cell lines tested and often was the only method that could produce indels. Transfection protocol lipofectamine 3000 transfection reagent hepg2 liver cancer cells complete growth medium component cat. A549 cells are an important model for drug metabolism and are also used as a host for gene transfection. Protocol for lipidmediated transient transfection in a549. Different cell lines were transfected with a gfp reporter vector using the indicated transfection reagents. Transfection protocol of adherent a549 cells 96well plate 1. Highest transfection efficiency in many cell types and formats e. If designing an experiment that involves transfection, ensure that setup coincides with a cell passage.
Selected citations for a549 transfection reagent used in the following publications. Cell line successful transfection dharmafect formulation lipid a lipid b lipid c lipid d a549 v v bxpc3 v du 145. 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. Gibco dmem with glutamax supplement 10566016 10% gibco fbs a3160401 proper culture techniques and procedures are an essential part of ensuring successful transfection. However, significant toxicity from lipofectamine 2000 was also reported in. I use 24well plates for transfections in triplicates. Lipofectamine 2000 transitx2 lipofectamine 2000 gapdh aha1 transfection reagent sirna target transitx2 dynamic delivery system continued 90 80 70 60 50 40 30 0 egfp positive % 20 10 a549 chok1 mdck lncap hmec 100 hep g2 pc12 nhdf transitx2 mirus bio was used to transfect plasmid dna encoding egfp into the indicated cell lines.
Transfecting plasmid dna into a549 cells using lipofectamine ltx. Lipofectamine 2000 transfection reagent dartmouth college. Maintain p19 cells undifferentiated in mem with 10% serum 7. Mirus lipofectamine 2000 and oligofectamine invitrogen. Methods the expression of mir16 in a549 cells was examined by quantitative realtime qrt. Count the cells using standard trypan blue exclusion. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Introducing the versatile reagent with the power to. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. 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.
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