The firstgeneration lipofection was a singlecomponent system, in which only the lipid nl. Figure 1 illustrates a typical, reagentbased transfection protocol. 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. 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. Optimization procedure for small interfering rna transfection in a 384well format. Ccl185 human lung carcinoma cells by fugene hd in 96 well plates. Commercially available a549 transfection reagent is provided by altogen biosystems link to manufacturer product page a549 transfection kit. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection.
Transfection efficiencies were assessed by luciferase activity rlu. Typically 100 ng and 500 ng of plasmid dna were transferred to each well of the 96well plate and 24well plates, respectively. Mirus lipofectamine 2000 and oligofectamine invitrogen. Transfection protocol of adherent a549 cells 96well plate 1.
Transfecting plasmid dna into a549 cells using lipofectamine. 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. Why is a549 so difficult to transfection researchgate. From expert transfection reagent developer molecular transfer inc. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Selected citations for a549 transfection reagent used in the following publications. 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. Transfecting plasmid dna into a549 cells using lipofectamine ltx reagent. Different cell lines were transfected with a gfp reporter vector using the indicated transfection reagents.
Transfection protocol transfection components component cat. 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. A transfection enhancer, the 3000 enhancer reagent 1. One can get over 4050% transfection efficiency with plasmid dna and over 85% with sirna. Transfection guide overview of transfection methods. The expression of type i and iii collagen in the tgf1. Highest transfection efficiency in many cell types and formats e. Scaling up or down lipofectamine 3000 reagent transfections. Gibco rpmi 1640 with glutamax supplement 61870036 10% gibco fbs a3160401 1. Transfection optimizationhow to get it right biocompare. Optimizing transfection lipofectamine 2000 cd reagent. Choosing the right transfection reagent for optimal efficiency. A549 cells are an important model for drug metabolism and are also used as a host for gene transfection. I have been working with a549 for quite a bit and i found lipofectamine 2000 or ltx to be the best transfection agents.
Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Highest transfection efficiency in many cell types and formats. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable. Enhanced transfection efficiency using lipofectamine 3000 reagent. 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. Improved delivery of cas9 proteingrna complexes using. Each reagent was tested with a range of transfection reagent. Lipofectamine 2000 reagent thermo fisher scientific. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Here, we report the results of a transfection trial with three promega reagents and another widely used transfection reagent. A549 transfection kit lung carcinoma, ccl185 altogen. For additional information on scaling your transfection reaction, see page 4.
Introduction lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Reagent, wv, was used along with the lipofectamine 3000 transfection reagent for all transfections. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. I have been transfecting a549 cells for long time but still could not get transfection efficiency more than 30%. Transfecting plasmid dna into a549 cells using lipofectamine 3000 reagent. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. The comparative utility of viromer red and lipofectamine. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. Fugene hd and lipofectamine 2000 should also work as i have seen them. Expifectamine 293 transfection kit protocol see page 7 to view a typical transfection procedure. 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.
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. Transfection conditions for expi293f cells for each 30ml transfection, use 7. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Refer to the cell lines database for a list of cell types successfully. Transfection guide overview of transfection methods promega.
Invitrogen lipofectamine 3000 transfection reagent 1. 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. It is a relatively modern method first described by felgner et al. Cell plating a549 cells were seeded from 90100% confluent culture the day before the transfection with the density 10,000 cellswell in 100. Scale your transfections up or down by proportionately adjusting the amounts of the reagents used. Figure 3 illustrates differences in expression and toxicity profiles for two common cell types, a549 and mdck cells. Lipofectamine 2000 transfection reagent dartmouth college. Protocol for lipidmediated transient transfection in a549 epithelial lung cell line.
Introducing the versatile reagent with the power to. Is there a better agent i can use for my a549 cell line. A549 lipofectamine 3000 lipofectamine 2000 fugene hd lipofectamine 2000 lipofectamine 3000 competitor 1 competitor 2 western blot, hepg2 gststat. Transfecting plasmid dna into a549 cells using lipofectamine ltx. Protocol for lipidmediated transient transfection in a549 epithelial. Cell line successful transfection dharmafect formulation lipid a lipid b lipid c lipid d a549 v v bxpc3 v du 145. How to maximize transfection efficiency in a549 cells. The reagent provides high transfection efficiency in many cells types and formats. Optimized conditions for successful transfection of human.
Transfection protocol lipofectamine 3000 transfection. I have used it both for stable and transient transfections. The kit is optimized to transfect sirna, mirna or dna plasmid following either a standard or reverse transfection. These cellspecific reagents offer maximum dna delivery.
A new transfection reagent formulated specifically for a549 cells. Comparative transfection efficiency of jetprime versus its main competitor. This reference provides a recommended procedure to transfect plasmid dna into human a549 lung carcinoma cells atcc, cat. Endofectin transfection reagent, cell lines genecopoeia. We also provide some tips for optimizing transfection conditions in. Cell lines were transfected with a firefly luciferaseexpressing plasmid. You can calculate this according to instructions in your reagent manual. The lipofectamine 2000 transfection experiment was carried out following the general protocol invitrogen lipofectamine 2000 technical manual, see supporting information for our particular procedure. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Transfection efficiencies lower than 50% were not reported in this table. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology.
42 552 1210 513 276 1512 389 33 640 231 858 895 1519 90 961 1007 564 1193 1167 991 1 1465 1444 328 1489 467 1128 266 1083 1184 1067 1199 1168 319 742 498