Transfection protocol lipofectamine 3000 transfection. A typical forward transfection protocol using transit2020 transfection reagent can be found here. Singlecell transfection by electroporation using an. Gibco rpmi 1640 with glutamax supplement 61870036 10% gibco fbs a3160401 1.
Highest transfection efficiency in many cell types and formats e. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1. A549 transfection kit lung carcinoma, ccl185 altogen. Dilute appropriate amount of lipofectamine 2000 by optimem i reduced serum medium to 50 l and vortex for a few seconds for siha and skov3 cells, dilute 1. For each transfection sample, prepare dnalipofectamine 2000 complexes as follows. Transfection protocol lipofectamine 3000 transfection reagent. 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. However with the variability of primary cell types, there are challenges for efficient delivery of dna or rna. Thee two most important factors in choosing transfecting reagent are. Transfection protocol lipofectamine 3000 transfection reagent a549 lung cancer cells complete growth medium component cat. Lipofectamine 2000 reagent thermo fisher scientific.
Rapid and highly efficient mammalian cell engineering via. Transfecting plasmid dna into a549 cells using lipofectamine ltx reagent. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. This reference provides general guidelines and procedures to transfect sirna into mammalian cells using lipofectamine 2000. All amounts and volumes are given on a per well basis. Transfect common cells efficiently and costeffectively for common cell types, lipofectamine 3000 reagent is more costeffective than other reagents due to its high concentration and low usage amount. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.
A549 cells were seeded into 6well plates and transfected with the mir415expressing vector or the control vector expressing a nonspecific mirnc using lipofectamine 2000 invitrogen, and were. A preoptimized a549 transfection kit is available from altogen biosystems, which includes. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Transfection protocol of adherent a549 cells 96well plate 1. Protocol for lipidmediated transient transfection in a549 epithelial lung cell line. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in a 96well format, and gfp expression was analyzed 48 hours posttransfection. 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 of cells with plasmid dna is well optimized, and the procedure is simple and quick. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Efficiently transfect difficulttotransfect cells lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of.
The day before transfection, trypsinize and count the cells. On the day of transfection just prior to preparing complexes, plate 1. Introduction lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Lipofectamine rnaimax is used only for small rnas, such as sirnas. Lipofectamine 2000 transfection reagent dartmouth college. Each reagent was tested with a range of transfection reagent. Improved delivery of cas9 proteingrna complexes using.
I use 24well plates for transfections in triplicates. Transfection efficiencies lower than 50% were not reported in this table. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Protocol for lipidmediated transient transfection in a549 epithelial. The day before transfection, dissociate cells that are 8090% confluent in a t75 flask. Prewarm 50ml of optimum stored in cold room at 4c 10min.
We also provide some tips for optimizing transfection conditions in cultured cells. For me, fugene and lipofectamine 3000 not 2000 worked the best, while the worst for me were pei and metafectene. One day prior to transfection, the cells were seeded in a 24well plate at a cell density of 12. Improved cell viabilitygentle on your cells, with low toxicity. Incubate at 37c for 4 to 6 hours for delicate cells or overnight for tough cells. At 48 hours posttransfection the a549 cells were analyzed by qrtpcr for gene expression levels.
Cell plating a549 cells were seeded from 90100% confluent culture the day before the transfection with the density 10,000 cellswell in 100. Lipofectamine 2000 transfection reagent from invitrogen. This allows the dna and the lipofectamine to form the complexes that will get the dna into the cells. To transfect cells with sirna, follow the protocol as described for dna but. Invitrogen lipofectamine 3000 transfection reagent. Invitrogen lipofectamine 3000 transfection reagent from. May 15, 2009 we found that 25 30% of cells were transfected with lipofectamine 2000 treatment, while at least 80% of the cells electroporated to the required extent m 0. Ccl185 human lung carcinoma cells by fugene hd in 96 well plates. Transfection guide overview of transfection methods promega. After 48 h post transfection, the cells were lysed and subjected to a genomic cleavage.
For reverse transfection b, a549 cells were plated after adding complexes following the protocol below. 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. Jul 24, 2007 lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Lipofectamine 3000 can be used to transfect a broad spectrum of cell line with nucleic acids. While immortalized cell lines traditionally have served as the common targets of transfection, primary cells are often preferred as they more closely resemble the invivo nature of tissues. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Here, we report the results of a transfection trial with three promega reagents and another widely used transfection reagent.
Cells should reach 5070% confluency at time of transfection. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. 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. Choosing the right transfection reagent for optimal efficiency. 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. Two component lipidbased proprietary formulation optimized for a549 cells transfection. Plate cells so they will be 7090% confluent at the time of transfection. Cells were transfected with a firefly luciferase encoding. Highest transfection efficiency in cells at 90% confluency.
Feb 18, 2016 upon identification of lipofectamine crisprmax as the best transfection reagent, we determined the functional activity of cas9 rnps by examining the times required for complexation of cas9 protein with grna cas9 rnps and cas9 rnps with lipofectamine crisprmax in a549, hek293, and hela cells. Invitrogen lipofectamine 3000 transfection reagent fisher. Plate cells for transfection only 1 day before the experiment. A549 cells are hypotriploid alveolar basal epithelial cells. This reference provides a recommended procedure to transfect plasmid dna into human a549 lung carcinoma cells atcc, cat. If designing an experiment that involves transfection, ensure that setup coincides with a cell passage. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Achieve superior transfection efficiency while maintaining low. Maintain p19 cells undifferentiated in mem with 10% serum 7. Higher densities can lead to spontaneous differentiation. Add the lipofectaminedna mixture to each well and gently swirl to mix. Transfecting plasmid dna into a549 cells using lipofectamine.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Add 6l of lipofectamine2000 from biostores and 100 l of. I spent about 5 months trying to optimize transfection protocols of 4 different cell lines including some very hard to transfect cells. This adenocarcinomic cell line is categorized as a nonsmall cell lung carcinoma nsclc, which tends to. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. Protocol for cell transfection of a549 xgal staining of a549 lung cancer cells 24 hours after transfection by fugenehd and competitor lipofectamine ltx and lipofectamine 2000 xgal staining of dms 53 human small cell lung carcinoma cells after transfection by fugenehd and reagents from competitor in 100% serum. For additional information on scaling your transfection reaction, see page 4. Versatileone reagent for dna, rna, and cotransfection. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format, and gfp expression was analyzed 48 hours posttransfection. After incubation, remove media and replace with fresh media. Transfecting plasmid dna into a549 cells using lipofectamine ltx.
Transfection protocol of adherent a549 cells 96well plate. If the transfected dna contains a fluorescent protein, check for transfection efficiency by viewing in a fluorescent microscope. A549 transfection reagent is tested functionally by transfection of a549 cells with a small interfering. The day before plate cells at 45x106p100 to get the following day 8090% confluency. Count the cells using standard trypan blue exclusion. For 96 well plate for me worked well plating approximately cells well transfecting them with 300ng dna and 0.
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