Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. 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. Transfection protocol lipofectamine 3000 transfection reagent a549 lung cancer cells complete growth medium component cat. Introducing the monster green fluorescent protein phmgfp vector using the transfast transfection reagent and the optimized transfection conditions resulted in the cell notes issue 7 2003 14 neuroscience transfecting a human neuroblastoma cell line with monster green fluorescent protein by natalie betz, ph. 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. Plate cells so they will be 7090% confluent at the time of transfection. 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. Prewarm 50ml of optimum stored in cold room at 4c 10min. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells. Add 6l of lipofectamine2000 from biostores and 100 l of. Invitrogen lipofectamine 2000 transfection reagent. Efficiently transfect difficulttotransfect cells lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of. A reverse transfection protocol was used to transfect mcf7 and hepg2 cells.
A549 transfection reagent is supplied in liquid form at a concentration of 0. Gibco rpmi 1640 with glutamax supplement 61870036 10% gibco fbs a3160401 1. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Is there a better agent i can use for my a549 cell line transfection. This protocol shows optimum transfection condition using hilymax in a549 cells. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas.
Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. Lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine 2000 reagent thermo fisher scientific. Commercially available a549 transfection reagent is provided by altogen biosystems link to manufacturer product page a549 transfection kit. Reverse transfection of slides printed with gelatindna microarrays materials gelatindna arrayed gaps slides above. Human lung adenocarcinoma a549 cells were transfected with synthetic. Fgfr3 or with control sirna using lipofectamine 2000 lipo2000 invitrogen.
Transient transfection of mammalian cells with lipofectamine. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Singlecell transfection by electroporation using an. Mdamb231 or a549 cells were transiently transfected with sivegfr3, sivegfc, or scramble control oligonucleotide purchased from dharmacon using lipofectamine 2000 invitrogen. Have you tried recovery step in your transfection protocol. Transfecting plasmid dna into a549 cells using lipofectamine. A549 transfection reagent is tested functionally by transfection of a549 cells with a small interfering.
Transfecting plasmid dna into a549 cells using lipofectamine 3000 reagent see all transfection protocols. The screening of transfection reagents was conducted in a 96well format. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. To transfect cells with sirna, follow the protocol as described for dna but. The media needs to be changed at least 1hr prior to transfection. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in a 96well format, and. To tranfect a549 cells in 24well plate, follow optimum condition for transfection and transfection procedure. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Introduction lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Transfection protocol lipofectamine 3000 transfection reagent. In a reverse transfection protocol, cells are added directly to a plate containing the. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna.
Feb 18, 2016 systematic design of experiments used for screening transfection reagents. Cell nuclei were stained with dapi after fixing and permeabilizing. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent. Highest transfection efficiency in cells at 90% confluency. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Thee two most important factors in choosing transfecting reagent are. Rapid and highly efficient mammalian cell engineering via.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. The 5ethynyl20deoxyuridine edu incorporation assay kit was purchased from ribobio and used after transfection. A549 and h1299 cells were transfected for 48 h using lipofectamine 2000 invitrogen. Improved delivery of cas9 proteingrna complexes using. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Fgfr3 silencing by sirna inhibits invasion of a549 cells ncbi. Highest transfection efficiency in many cell types and formats e. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. Seeding cells for transfection the day before transfection, dissociate cells that are 8090% confluent in a t75 flask.
Improved cell viabilitygentle on your cells, with low toxicity. Versatileone reagent for dna, rna, and cotransfection. Transfecting a human neuroblastoma cell line with monster. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. A sample protocol is listed here for transfection experiments performed in 6well plates. The day before transfection, dissociate cells that are 8090% confluent in a t75 flask. General procedure for transfection of mammalian cells with. Count the cells using standard trypan blue exclusion. We also tested other adherent cell lines cos7, nih 3t3, hela, a549 and found them to give transfection efficiencies of 3050% of hek293t cells. Transient plasmid dna transfection into hela and hacat cells. Transfection guide overview of transfection methods promega. The protocol involves mixing dna with calcium chloride, adding this mixture in a controlled manner to.
May 15, 2009 singlecell transfection following electroporation. Invitrogen lipofectamine 3000 transfection reagent. A549 cells are an important model for drug metabolism and are also used as a host for gene transfection. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. This reference provides a recommended procedure to transfect plasmid dna into human a549 lung carcinoma cells atcc, cat. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. 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 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. Transfection protocol lipofectamine 3000 transfection. If designing an experiment that involves transfection, ensure that setup coincides with a cell passage. Stat3induced upregulation of circccdc66 facilitates the. Using this scep method, we have successfully delivered pegfpc2 plasmid into a549 cells with an extracellular buffer plasmidfilled capillary having a pulled tip with a 2 m opening. One day prior to transfection, the cells were seeded in a 24well plate at a cell density of 12. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. For additional information on scaling your transfection reaction, see page 4. 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. Fgfr3 silencing by sirna inhibits invasion of a549 cells.
The day before plate cells at 45x106p100 to get the following day 8090% confluency. Is there a better agent i can use for my a549 cell line. Higher densities can lead to spontaneous differentiation. Invitrogen lipofectamine 3000 transfection reagent fisher. Plate cells for transfection only 1 day before the experiment. Supplemental data further evidence for expression and. Scaling up or down lipofectamine 3000 reagent transfections. When using the other vessel, refer to table 2 and adjust the amounts of cells, medium, dna and hilymax in proportion to the relative surface area. Maintain p19 cells undifferentiated in mem with 10% serum 7. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Brightfield image of a549 cells in culture, prior to transfection. A549 transfection kit lung carcinoma, ccl185 altogen. I use 24well plates for transfections in triplicates.
Two component lipidbased proprietary formulation optimized for a549 cells transfection. Superior performance for co transfection of sirna and plasmid dna. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. I would suggest you use lipofectamine 2000 or ltx and optimize cell confluency for best results. Mar 27, 2007 lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Lipofectamine 2000 transfection reagent from invitrogen. Cells should be plated on collagencoated dishes, as for normal propagation.
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