Lipofectamine 2000 cd reagent thermo fisher scientific. Transfection protocol of adherent a549 cells 96well plate. Cell plating a549 cells were seeded from 90100% confluent culture the day before the transfection with the density 10,000 cells well in 100. Table 2 examines transfection efficiency and cell viability under optimal conditions for each reagent 24 hours after transfection. Protocol for lipidmediated transient transfection in a549 epithelial. Transfection efficiency and protein expression using geneart crispr nuclease vector. Transfection protocol lipofectamine 3000 transfection. These cell specific reagents offer maximum dna delivery. We observed a high batchtobatch variation in performance of the lipofectamine 2000 transfection reagent.
Recombinant bcl2 shrnas expression vector were transfected into a549 cells with lipofectamine 2000. C2c12 transfection reagent mouse myoblast cells invivo. A375 cells were seeded from 90100% confluent culture the day before transfection with the density 15,000 cells per well in 100. Rapid and highly efficient mammalian cell engineering via. Cells were also assayed for cell viability and compared with a no transfection control for each cell line. Plate cells so they will be 7090% confluent at the time of transfection. 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. In a549 cells transfected with hsp27interfering plasmid, the. A549 cells are human alveolar basal epithelial cells that grow adherently as a monolayer in vitro and serve as suitable transfection hosts. Jun 01, 2004 abstract lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Trials of transfection in eukaryotic cells are essential tools for the study of gene and protein function. I found lipofectamine and geneporter2 might not only damage a549 also have worse tranfection efficiecy, so i used jetpei to transfection. They have been used in a wide range of research fields. Fast, simple, easytofollow protocol onestep process.
On the day of transfection, 500 ng cas9 protein and 120 ng hprt1 grna were transfected with either lipofectamine 2000 lf2k, lipofectamine 3000 lf3k, lipofectamine messengermax, lipofectamine rnaimax, turbofect or xfect transfection reagent. Protocols for transfecting the most common cell lines with x. These cells could be used for biomedical research and cell biology, especially of mammalian myoblast cells. The same batch of the transfection reagent should be used for the whole experiment.
For stable transfection, the cells were transfected using lipofectamine 2000. Ccl185 human lung carcinoma cells by fugene hd in 96 well plates. Transfection protocol lipofectamine 3000 transfection reagent a549 lung cancer cells complete growth medium component cat. Transfection guide overview of transfection methods promega. Expression and function of gsta1 in lung cancer cells. While lipofectamine 2000 reagent demonstrated transfection efficiencies only slightly lower those of viafect reagent, it was considerably more toxic to hela cells at all tested ratios of reagent to dna. Invitrogen lipofectamine 3000 transfection reagent. Transfection guide overview of transfection methods. To transfect cells with sirna, follow the protocol as described for dna but. Stem cell transfection gene knockdown lipofectamine 2000 keratinocytes mdamb453 neuro2a shsy5y sknmc au565 bxpc3 cos7 hela hct 116 mcf7 mdamb231 pc3 transitx2 dynamic delivery system a549 aspc1 bhk21 bj bt20 caco2 c2c12 cfpac1 chok1 ditnc1 du145 hep g2 hcc1143 hcc38 hek 293 hmec. A549 after 72 h following double transfection of lipofectamine 2000.
After overnight incubation, cells were cultured for an additional 48 h. Phosphorylationdependent feedback inhibition of rigi by dapk1. Introduction lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. This adenocarcinomic cell line is categorized as a nonsmall cell lung carcinoma nsclc, which tends to. Bcl2 small hairpin rnas enhance radiationinduced apoptosis. Xgal staining of a549 lung cancer cells 24 hours after.
Luciferase activity was measured post transfection of a luciferase coding plasmid using different chemical transfection reagents, transitx2, lipofectamine 2000, and lipofectamine 3000. We used this kit for efficient transfection of cell lines such as hl60, mdamb231, and raw 264. Advanced transfection with lipofectamine 2000 reagent. L growth medium for a single well of a 24well plate. Suppression of human lung cancer cell proliferation and metastasis. Protocol for a375 cell transfection by fugene hd with. The protocol involves mixing dna with calcium chloride, adding this mixture in a controlled manner to. Transfecting plasmid dna into a549 cells using lipofectamine. 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. Lipofectamine transfection of c2c12 cells bridges lab. High gene transfection efficiency of fluorinedoped carbon. L lipofectamine 2000 invitrogen according to the manufacturers instructions.
Dnain cell specific transfection reagents are a line of transfection products developed by the lead members of the scientific team that invented the lipofectamine and lipofectamine 2000. Lipofectmax transfection reagent has been tested to work the same efficiency as lipofectamine 2000 reagent, and used for the transfection of both. Plate adherent cells one day before transfection so that cells are approximately 80% confluent on the day of transfection. Ac a549 cells transfected with the indicated sirnas were infected with. For each transfection sample, prepare complexes as follows. At weight ratio transfection reagentegfp plasmid of 2, the transfection efficiency of fcds achieves about 92% in hek 293t cells, 85% in cos7 cells, 81% in a549 cells, 73% in b16f10 cells, 58% in nih3t3 cells and 45% in hepg2 cells, which is much higher than that of lipofectamine 2000 in hek 293t cells 65%, cos7 cells 33%, a549 cells. Total rna was extracted 24 hours after transfection, and total cell protein were extracted 48 or 72 hours after transfection. A549 cells, u2 os cells, neuro2a n2a cells were purchased from atcc. Composites of malonic acid diamides and phospholipids. Viafect transfection reagent transfection assay e4981. Wash the cells once with serumfree growth medium without antibiotics, and seed cells at a density of 23 x 106 cells per well in 0.
Sep 19, 2018 figure 3 illustrates differences in expression and toxicity profiles for two common cell types, a549 and mdck cells. A549 cells are positive for keratin by immunoperoxidase staining. Dharmafect volumes per well represent guidelines and need to be optimized. The reagent provides high transfection efficiency in many cells types and formats. Help on transfecting mcf7 and hct 116 cells transfection.
Transfected cells were screened in 800 mgml g418 screening medium, and after stable transfection, silencing was examined. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Lipofectamine2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 reagent thermo fisher scientific. Micrornaa b targets pten to induce resistance to cisplatin. Lipofectamine 3000 transfection kit thermo fisher bioz. Subsequently, cell viability, growth and apoptosis of a459 cells in the mir. The amount of lipofectamine 2000 should be changed in proportion to the culture medium. Reverse transfection protocol for dna in 96well plates using transitx2. The following is a general protocol for use of dharmafect transfection reagents to deliver sirna into cultured mammalian cells. A549 cells are a cell line derived from an adenocarcinoma, originally developed in 1972 by removing and culturing lung tumor. On the day of transfection, prepare a single cell suspension from stock cells. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul. The other transfection method was that the recombinant retroviral supernatant was added into.
The viability of the transfected cells were assessed by mtt. When commercial pulmonary surfactant infasurf was added in the transfection medium, the gene silencing effect of sirna in cells transfected with lipofectamine 2000 was completely. Transfecting primary cells in suspension transfection. In a reverse transfection protocol, cells are added directly to a plate containing the. Transfection protocol of adherent a549 cells 96well plate 1. Regarding cytotoxicity of hct116 cells, they at confluency. If cells are not at the right confluence, do not wait until the next day to perform transfection, as this can significantly affect transfection efficiency. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Liposome mediated recombinant plasmids were transfected into bmscs cells in accordance with the manual of lipofectaminetm 2000 kit invitrogen, usa. In a headtohead comparison study, dnain a549 reagent consistently outperformed lipofectamine 2000 and lipofectamine 3000 transfection reagents. Gfp expression in a375 melanoma cells after transfection. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Add the lipofectamine dna mixture to each well and gently swirl to mix.
A reverse transfection protocol was used to transfect mcf7 and hepg2 cells. The wounds were carefully created manually on the monolayers using sterile. 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. Gibco rpmi 1640 with glutamax supplement 61870036 10% gibco fbs a3160401 1. Following incubation for 24 h, the cells were transfected with satb1sirna1, satb1sirna2 and satb1sirnan plasmid in serumfree medium using lipofectamine 2000 reagent invitrogen, carlsbad, ca, usa according to the manufacturers instructions. Improved delivery of cas9 proteingrna complexes using. Cten induces epithelialmesenchymal transition emt and. One day prior to transfection, the cells were seeded in a 24well plate at a cell density of 12. The lipid mixtures with the fluid cationic lipid oh4 and dope have shown comparable transfection efficiency with lipofectamine 2000.
The use of enhanced green fluorescent proteinexpressing cell. Aug 04, 2006 i have used 3 kinds of transfection reagents lipofectamine, geneporter2, jetpei to test which one was better for a549 cell. Lipofectamine 2000 was also reported in u2os cells. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
The pcmvvector, pcmvcten, controlshrna, and ctenshrna were transfected into a549 and ncih1299 cells by lipofectamine 2000. Feb 22, 20 cells in a 6 well dish, plated and at 70% confluence. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. From expert transfection reagent developer molecular transfer inc. Incubate at 37c for 4 to 6 hours for delicate cells or overnight for tough cells. Owing to its ability for rapid differentiation that can be easily modulated by treatment with proteins, the c2c12 cell line has proven to be a reliable transfection host for the study of muscle cells. This reference provides a recommended procedure to transfect plasmid dna into human a549 lung carcinoma cells atcc, cat. The expression and location of recombinants were detected by fv laser scanning microscope.
From the luciferase activities and consistencies of data using different transfection reagents including genejammer, effectine, wellfect, lipofectamine, what i can say from the data i observed, the efficiency of transfection is much more better in hct116 cells using lipofectamine 2000. Commercially available a549 transfection reagent is provided by altogen biosystems link to manufacturer product page a549 transfection kit. The a549 cells were transcendtransfected with tob1sirna. Three doublestrand dna oligornas sirnas were synthesized prior to being transfected into a549 cells with lipofectamine 2000, and then the most efficient sirna was selected. You can calculate this according to instructions in your reagent manual. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Cell proliferation assay cell proliferation assay was determined by cell. A549 cells are hypotriploid alveolar basal epithelial cells. The mechanism of lipofectamine 2000 mediated transmembrane. Transfection optimizationhow to get it right biocompare. Protocols for transfecting the most common cell lines with. Maintain p19 cells undifferentiated in mem with 10% serum 7. Expression of the bcl2 protein was assayed using western blot in a549 cells.
A375 cells were at approximately 80% confluence on a day of transfection. In the present study, we screened a large set of transfection reagents using relatively easy and hardtotransfectcelllines and identi. Transfection protocol lipofectamine 3000 transfection reagent. Suspension cells can be plated the day of transfection. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng. Inhibition of interferon signaling by dengue virus pnas. 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 lipid. Original article study on the effect of bmscsegfptk as. From pulmonary surfactant, synthetic kl4 peptide as. A549 cells were transiently transfected with the recombinants using lipofectamine 2000 and the transfection efficiency was confirmed through gfp signal. A new transfection reagent formulated specifically for a549 cells. We found that expression in human a549 cells of the dengue virus nonstructural proteins. The initial seeding density of a549 cells 24 h before transfection. Transfection efficiencies lower than 50% were not reported in this table.
Effect of silencing satb1 on proliferation, invasion and. Lipofectmax transfection reagent is a lipidbased transfection reagent that forms a complex with dna or rna, and transports the complex into a variety of adherent and suspension cell lines. A549 lung carcinoma cells were transfected with mir. Expression of the gsta1 gene in the transfected cells was determined by realtime pcr and western blotting. A549 cells were transfected by using lipofectamine 2000 invitrogen as speci. Microrna7 inhibits the growth of human nonsmall cell lung.
It mediated sirna transfection effectively in vitro on human lung epithelial cells, a549 cells, and beas2b cells, which was comparable to lipofectamine 2000. After incubation, remove media and replace with fresh media. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. If the transfected dna contains a fluorescent protein, check for transfection efficiency by viewing in a fluorescent microscope. A549 and h446 cells had been seeded in plates with 12 wells for exactly 1 day to reach 20% to 30% confluence and then gone through transfection with lipofectamine 2000according to manufacturer invitrogens manual. Several cell and application specific dnain transfection reagents also have been developed. An example of high efficiency forward and reverse transfection of a549 cells using. A preoptimized a549 transfection kit is available from. Feb 18, 2016 identification of lipofectamine crisprmax. Reverse transfection on cell arrays for high content.
Lipofectamine 2000 was used in the current study as the protocol was. A549 cells are an important model for drug metabolism and are also used as a host for gene transfection. Choosing the right transfection reagent for optimal efficiency. Lipofectamine 2000 transfection reagent sirna rnai mirna. Achieve superior transfection efficiency while maintaining low. In the first attempt, the exact protocol by the manufacturer was foll. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Nanoparticlemediated gene silencing for sensitization of. Sitedirected mutagenesis and protein expression of abca3.
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