Lipofectamine 2000 5,000 cells well 10,000 cells well 25,000 cells well normalized values % 140 120 100 80 60 40 20 0. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common cells e. After 4 to 6 h of transfection, the medium was replaced with fresh medium. Enhanced green fluorescent protein genes pegfp were introduced into cos 7 cells using a cationic lipidbased reagent, lipofectamine 2000. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Add 25 l of the diluted lipofectamine 2000 from step 3 to each well containing diluted dna from step 2, mix gently, and incubate at room temperature for 20 min to allow dna lipofectamine 2000 complexes to form. Dilute in complete growth medium to 5 x 104 cellsml 2. Cell surface proteinprotein binding on cos7 cells europe. For ip, large cell numbers approximately 2 x 107 were required. Assay for phenotype or target gene expression 48 72 hours after transfection 1.
Lipofectamine transient transfection of cos7 cells to analyze. Cos7 cells in microchannels with lipofectamine 2000. Download altogen biosystems cos7 transfection protocol. The medium for cos7 cells in a screwcapped flask 25 cm2 was replaced by phenol redfree mem nissui pharmaceutical co.
Jan 05, 2015 transfect the cells with the plasmid encoding the extracellular protein fused to the human igg fc. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. L3000001 lipofectamine 3000 transfection reagent sparshbio. Add the dna lipofectamine 2000 complexes 50 l directly to each well of the plates containing cells and mix. Lack of evidence for at1rb2r heterodimerization in cos7. Transfection of the cell lines cos7 and p815 using metafectene. Mammalian cells cos7 a division of gene therapy systems, inc. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. The cells were then transfected with lipofectamine 2000 invitrogen. Lipofectamine 2000 transfection reagent 11668027 0. The addition of antibiotics to media during transfection may result in cell death, and has not been tested for cos7 cells. Follow these important guidelines when transfecting dna into cos7 cells using lipofectamine ltx reagent. Transfection was performed in triplicate 6well plate 4 times.
Briefly, cos7 cells were seeded on to 24 well, tissue culture treated plates at a density of 5 x 104 cellscm 2. In cos7 cells, expression of human ctgf induced an altered cell morphology. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. To transfect cells in different tissue culture formats, vary the amounts of lipofectamine 2000, nucleic acid, cells, and medium used in proportion to the relative surface area, as shown in the table. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Cell culture and transfection 293t and cos7 cells were maintained in dul beccos from bme 301 at new jersey institute of technology. Cos7 cells were grown in dmem supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. Transfecting plasmid dna into cos7 cells using lipofectamine. The plasmid dnas of mirna and blg expression constructs were mixed at a molar ratio of 1. Increased transfection efficiency by the directed transport, especially for low amounts of nucleic acids high transfection rates for adherent mammalian cell lines and primary cell cultures suspension cells and cells from other organisms also successfully transfected but need to be immobilized mild treatment of cells.
Cos7 cells, seeded in 12well plates, were transiently transfected with wtnprb, mutant nprbs, or empty vector. Photoacoustic transfection of dna encoding gfp scientific. Cells were incubated at 37 c in humidified 95% air and 5% co 2 atmosphere, they were subcultured before they reach 90% confluency. Cadmium exposure induces expression of the hoxb8 gene in cos. Efficiently transfect difficulttotransfect cells lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. A cellpenetrating helical polymer for sirna delivery to. Electroporation as a cell transfection method for high. This reference provides a recommended procedure to transfect plasmid dna into cos7 monkey kidney fibroblasts atcc cat. Cv1, cos1, or cos7 cells are widely used as host cell lines because they do not carry any. Transfection efficiency in cos7 cells transfection number of positive cells reagent amount of dna ratio reagent absolute. Monolayers of transfected cells were incubated in serumfree growth medium supplemented with 10 mm hepes, ph 7. The maxcyte stx scalable transfection system is a benchtop instrument that can transfect small scale 5e5 cellsseconds to hts scale. Enhanced green fluorescent protein genes pegfp were introduced into cos7 cells using a cationic lipidbased reagent, lipofectamine 2000. Six different recombinant plasmids were prepared with the endofree plasmid maxi kit and then used to transfect mef, c6 and cos7 cells, independently, by using lipofectamine 2000 reagent.
Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2. An easy protocol for onchip transfection of cos7 cells with. Nektaria andronikou, xin yu, jia wei, mahalakshmi sridharan. Lipofectamine 2000 reagent fugene hd reagent 120% 10 nm 30 nm 80% 20% n e 000 000 n 000e 000 x n 000 000 x 100% % remaining expression 40% 60% 0% 143b hek 293 mcf7 18% lipofectamine 2000 lipofectamine 3000 52% methods figure 1.
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. Increased transfection efficiency by the directed transport, especially for low amounts of nucleic acids high transfection rates for adherent mammalian cell lines and primary cell cultures suspension cells and cells from other organisms also successfully transfected but. Plate cells so they will be 7090% confluent at the time of transfection. Lipofectamine 2000 transfection reagent dartmouth college. The trusted, simple, broadspectrum transfection reagent for most cell lines. This protocol shows optimum transfection condition using hilymax in cos 7 cells. Though the study focused on lipofectamine 2000 and cos7 cells, this research method could be. Has anyone used lipofectamine 3000 for plasmid transfection. Dna numb er relative amount metafectene 2 g 71 appr. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. For the dissociation kinetics analysis of the vvd variants, cos7 cells were plated at 10. Cells were transiently transfected with lipofectamine 2000 life technologies according to the manufacturers protocol. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf.
The products included in this piece are our most robust transfection reagents that provide solutions to both common and hardtotransfect cell types, promoting effective. With automated, highthroughput systems, a complexing volume of 50 l is recommended for transfections in 96well plates. The hoxb8 and negativecontrol sirnas were individually transfected into cos7 cells grown in 6well culture plates 50% confluence using lipofectamine 2000 invitrogen according to the manufacturers instructions. Cellspecific transfection protocols thermo fisher scientific us.
Figure 7 shows the gfp expression in cos7 cells 24 h after transfection with lipofectamine 2000 incubation time of 4 h or with the pa waves generated with a 30 ps laser exposure time of 10. Investigation of neural stem cellspecific regulatory. In this study, a microchip made of polydimethylsiloxane pdms and glass was fabricated for continuous cell culture and transfection. Expression of the target gene in cells transfected with recombinant plasmid. The day after transfection they were seeded onto 6well plates. Enhanced green fluorescent protein genes pegfp were introduced into cos7 cells using a cationic lipid based reagent, lipofectamine 2000. The optimal concentration of lipofectamine 2000 was also studied. The amenability to a wide variety of transfection techniques, the high efficiency and accuracy in the. Cadmium exposure induces expression of the hoxb8 gene in. Cells were cultured for 12 to 16 h before transfection and transfected at.
Apr 25, 2017 a synj2bp overexpression assay in cos 7 cells. In vitro studies of magnetically enhanced transfection in cos. Enhancement of the efficiency of peiliposome transfection. Lipofectamine 2000 right panel around 5% efficiency. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. I would like to transfect 293t cells with lipofectamine 3000. An easy protocol for onchip transfection of cos7 cells with a. Supplemental data human prolinerich nuclear receptor.
Lipofectamine 2000 and lipofectamine 3000 were used to transfect u2os and. If you wish to use antibiotics during transfection, test your conditions thoroughly. A focused dna microarray analysis of estrogenresponsive. However, they have been frozen in a state of suspended high efficiency. Supporting information genetically engineered photo. C seeding 12wells, 2x105 cells well, prior to transfection, incubate overnight 8090 % confluency. Lipofectamine transfection reagents shipped at ambient. Cos7 monkey kidney cells were cultured in roswell park memorial institute 1640 medium, supplemented with 10% fetal bovine serum, 1% lglutamine and 1% penicillinstreptomycin complete rpmi. Twentyfour hours after transfection, cos7 were assayed for insitu. 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. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent.
Cos7 cells, an african green monkey kidney cell line atcc were transfected with pvax1lacz invitrogen using lipofectamine 2000 transfection reagent invitrogen. Transfect the cells with the plasmid encoding the extracellular protein fused to the human igg fc. Prepare 6 well plates by adding bakedsterile 22x22 1. Huh7, human hepatocellular carcinoma, plasmid dna, lipofectamine 3000. Lipofectamine transient transfection of cos7 cells to. Aug 07, 2009 in this study, a microchip made of polydimethylsiloxane pdms and glass was fabricated for continuous cell culture and transfection. To simplify the transfection optimization process, we have created a. Cell reagents required for the assay are cos7 cells transiently transfected with 2 plasmids using lipofectamine 2000. Figures and data in proteomic mapping of cytosolfacing outer. Transient transfection of cos7 and hekigf1r cells was performed using lipofectamine life technologies, inc.
Lipofectamine 2000 and lipofectamine 3000 were used to transfect u2os and hepg2 cells in a 12well format. Invitrogen lipofectamine 3000 transfection reagent 1. To express the proteins in cultured cells, a variety of plasmids for gal3 deletion mutants were transfected into cos7 and bt549 cells using lipofectamine 2000 reagent invitrogen. Cell culture and transfection 293t and cos7 cells were.
Cos7 cells 3 x 107 or hela cells 1 x 107 were transiently transfected with the indicated plasmids using the calcium phosphate method or lipofectamine 2000 invitrogen, respectively, as previously described ishigaki et al. A comparison showing transfection efficiency of polyjet reagent vs. After 24 hours, the cells were transfected using lipofectaminetm 2000 according to manufacturers protocol. For downstream experiments, solely rotifect plus was used. Rotifect plus was at least as efficient as lipofectamine. Figure 3 transfection of difficulttotransfect cells with lipofectamine 2000. Cos 7 cells were transfected with synj2bpv5 and mcherrykdel an er marker using lipofectamine 2000. Incubate cells for days at 37c amount 96well 24well 6well dnawell 100 ng 500 ng 2500 ng lipofectamine 2000 reagent well 0. The cos7 cells were cotransfected with plasmids by using lipofectaminetm 2000 invitrogen co.
Lipofectamine 2000 reagent thermo fisher scientific. Cos7 cells were transfected with synj2bpv5 and mcherrykdel an er marker using lipofectamine 2000. Lipofectamine 2000 5,000 cellswell 10,000 cellswell 25,000 cellswell normalized values % 140 120 100 80 60 40 20 0. Cos7 transfection reagent kidney cells, crl1651 altogen. Characterizationofthenuclearimportpathwaysofgalectin3. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Highest transfection efficiency in many cell types and formats e. Engineered pairs of distinct photoswitches for optogenetic. Pdf an easy protocol for onchip transfection of cos7. Cos 7 cells were transfected by magnetofectins mm0. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Heterozygous mutations in natriuretic peptide receptorb. This protocol shows optimum transfection condition using hilymax in cos7 cells. Lipofectamine 2000 reagent fugene hd reagent 120% 10 nm 30 nm 80% 20% n e 000 000 n 000e 000 x n 000 000 x 100% % remaining expression 40% 60% 0% 143b hek 293 mcf 7 18% lipofectamine 2000 lipofectamine 3000 52% methods figure 1.
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