Is there a better agent i can use for my a549 cell line. Transfection protocol of adherent a549 cells 96well plate 1. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Transfection guide overview of transfection methods. Transfection protocol lipofectamine 3000 transfection. Fugene hd and lipofectamine 2000 should also work as i have seen them. Refer to the cell lines database for a list of cell types successfully. 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. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Cell line successful transfection dharmafect formulation lipid a lipid b lipid c lipid d a549 v v bxpc3 v du 145. Improved delivery of cas9 proteingrna complexes using. Transfection guide overview of transfection methods promega. This reference provides a recommended procedure to transfect plasmid dna into human a549 lung carcinoma cells atcc, cat. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology.
Transfection conditions for expi293f cells for each 30ml transfection, use 7. Scale your transfections up or down by proportionately adjusting the amounts of the reagents used. For additional information on scaling your transfection reaction, see page 4. One can get over 4050% transfection efficiency with plasmid dna and over 85% with sirna. Introducing the versatile reagent with the power to. Here, we report the results of a transfection trial with three promega reagents and another widely used transfection reagent. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. From expert transfection reagent developer molecular transfer inc. Transfecting plasmid dna into a549 cells using lipofectamine ltx reagent. Reverse transfection for suspension cells andor high throughput applications, a reverse transfection protocol where freshly passaged cells are added to preplated transfection complexes is ideal as it reduces handson time for the end user. Figure 3 illustrates differences in expression and toxicity profiles for two common cell types, a549 and mdck cells. Transfecting plasmid dna into a549 cells using lipofectamine 3000 reagent. Mirus lipofectamine 2000 and oligofectamine invitrogen.
Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Scaling up or down lipofectamine 3000 reagent transfections. Each reagent was tested with a range of transfection reagent. Transfecting plasmid dna into a549 cells using lipofectamine ltx. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. Transfection efficiencies lower than 50% were not reported in this table. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Lipofectamine 2000 reagent thermo fisher scientific. Protocol for lipidmediated transient transfection in a549 epithelial lung cell line. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. We also provide some tips for optimizing transfection conditions in. Ccl185 human lung carcinoma cells by fugene hd in 96 well plates. A transfection enhancer, the 3000 enhancer reagent 1.
Highest transfection efficiency in many cell types and formats. The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine 2000 transfection reagent dartmouth college. The comparative utility of viromer red and lipofectamine. Invitrogen lipofectamine 3000 transfection reagent 1. Singlecell transfection by electroporation using an. I have used lipofectamine and fugene for plasmid transfection. Endofectin transfection reagent, cell lines genecopoeia.
At 72 hours post transfection the cells were analyzed by western blot for protein expression levels normalized by total protein, 10 g of total protein loaded per each well. Optimization procedure for small interfering rna transfection in a 384well format. To transfect cells with sirna, follow the protocol as described for dna but. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Introduction lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. 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. The lipofectamine 2000 transfection experiment was carried out following the general protocol invitrogen lipofectamine 2000 technical manual, see supporting information for our particular procedure. Transfection protocol lipofectamine 3000 transfection reagent a549 lung cancer cells complete growth medium component cat. Transfecting plasmid dna into a549 cells using lipofectamine. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Optimizing transfection lipofectamine 2000 cd reagent. Choosing the right transfection reagent for optimal efficiency. Optimized conditions for successful transfection of human. Figure 1 illustrates a typical, reagentbased transfection protocol. Transfection efficiencies were assessed by luciferase activity rlu.
Expifectamine 293 transfection kit protocol see page 7 to view a typical transfection procedure. 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. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. The firstgeneration lipofection was a singlecomponent system, in which only the lipid nl. Transfection protocol transfection components component cat. Transfection optimizationhow to get it right biocompare. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. 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. Selected citations for a549 transfection reagent used in the following publications. Cell plating a549 cells were seeded from 90100% confluent culture the day before the transfection with the density 10,000 cellswell in 100. I have been transfecting a549 cells for long time but still could not get transfection efficiency more than 30%. It is a relatively modern method first described by felgner et al.
Why is a549 so difficult to transfection researchgate. Gibco rpmi 1640 with glutamax supplement 61870036 10% gibco fbs a3160401 1. Comparative transfection efficiency of jetprime versus its main competitor. How to maximize transfection efficiency in a549 cells. The expression of type i and iii collagen in the tgf1. A new transfection reagent formulated specifically for a549 cells. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. Expi293f expression system kit thermo fisher scientific.
Reagent, wv, was used along with the lipofectamine 3000 transfection reagent for all transfections. A549 cells are an important model for drug metabolism and are also used as a host for gene transfection. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. Different cell lines were transfected with a gfp reporter vector using the indicated transfection reagents. Protocol for lipidmediated transient transfection in a549 epithelial. Cell lines were transfected with a firefly luciferaseexpressing plasmid. I have used it both for stable and transient transfections. Lipofection has become more common than the capo 4 precipitation, because it is just as simple to use but frequently provides a much better transfection efficiency.
A549 lipofectamine 3000 lipofectamine 2000 fugene hd lipofectamine 2000 lipofectamine 3000 competitor 1 competitor 2 western blot, hepg2 gststat. Commercially available a549 transfection reagent is provided by altogen biosystems link to manufacturer product page a549 transfection kit. 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. You can calculate this according to instructions in your reagent manual. Enhanced transfection efficiency using lipofectamine 3000 reagent. These cellspecific reagents offer maximum dna delivery. Typically 100 ng and 500 ng of plasmid dna were transferred to each well of the 96well plate and 24well plates, respectively.
The kit is optimized to transfect sirna, mirna or dna plasmid following either a standard or reverse transfection. A549 transfection kit lung carcinoma, ccl185 altogen. Cells were transfected with a firefly luciferase encoding plasmid dna 0. I have been working with a549 for quite a bit and i found lipofectamine 2000 or ltx to be the best transfection agents. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable. Transfection protocol of adherent a549 cells 96well plate.
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