Transfection of hek293 cells with lipofectamine 2000 manual pdf

These conditions give 70% transfection efficiency with only very moderate cell death. Lipofectamine 2000 reagent thermo fisher scientific. Comparative transfection of dna into primary and transformed. Lipofectamine 2000 transfection reagent from invitrogen. Lipofectamine rnaimax has a broad peak of activity. Transfection of hek293ebna1 cells in suspension with. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng. Transfection guide overview of transfection methods. Would anyone have an efficient protocol for transfection of. 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.

Optimized conditions for successful transfection of human. Fast and efficient transfection of mouse embryonic stem cells. Fast and efficient transfection of mouse embryonic stem. However, efforts are still needed to optimize production parameters in order to maximize volumetric pr. Plate cells so they will be 7090% confluent at the time of transfection. The reagent provides high transfection efficiency in many cells types and formats. Genetic transfection an overview sciencedirect topics. Transfection of hek and bj cells with lipofectamine 2000. Plate adherent cells one day before transfection so that cells are approximately 80% confluent on the day of transfection. Oligo into mammalian cells using lipofectamine 2000. If you let them reach confluency, even if this happened a few passages ago, the transfection efficiency will be significantly diminished. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. For additional information, and protocols on transfecting suspension cells refer to the manual at. Reverse transfections of adherent cells in 96well plates.

Lipofectamine 3000 is the best choice for transfection of chok1 and hek293 with pcdh while turbofect is preferably used in transfecting these cell lines with pegfpn1 tab. If designing an experiment that involves transfection, ensure that setup coincides with a cell passage. Hek 293 cells are useful for many transfection experiments, particularly the propagation of adenoviralbased and retroviralbased vectors. This reference provides a recommended procedure to transfect plasmid dna into hek 293, human embryonic kidney cells atcc no. Evaluation of transfection methods for transient gene. In particular, the linear isoform of pei is more effective for transfecting cells in suspension. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. Serumfree medium is necessary for successful dna lipofectamine 2000. To transfect cells with sirna, follow the protocol as. Transfection of hek293ebna1 cells in suspension with linear.

We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. Change medium within 18hours remove the old medium, add 10 ml fresh growth medium. Dharmafect transfection reagentssirna transfection. I use lipofectamine 3000 to transfect hek 293 cells in 6 wells plates. Transfection amounts component 96well 24well 6well final mrna per well 100 ng 500 ng 2500 ng final lipofectamine. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection. 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.

Feb 08, 2010 with the exception of hek 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering dna into the cells as determined by cell proliferation and gfp expression, respectively. Transfection protocol lipofectamine 3000 transfection reagent. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. For suspension cultures, transfect cells at a density of 0. The mechanism of lipofectamine 2000 mediated transmembrane.

Do not add antibiotics to media during transfection as this causes cell death. 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. May 28, 2010 the transfection efficiency of lipofectamine 2000 pires2egfp into hek293 cells was 2. Perform the transfection in the late afternoon because the transfection mix should only be incubated with the cells for 1215 hours. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. In my experience lipofectamine works like a charm in many cells, including hek293. I agree with parvoman that one of the main considerations is how well the cells are growing. Gene expression by largescale transfection of mammalian cells is becoming an established technology for the fast production of milligram and even gram amounts of recombinant proteins rproteins.

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. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. One day prior to transfection, the cells were seeded in a 24well plate at a cell density of 12. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure.

Cells were also assayed for cell viability and compared with a no transfection control for each cell line. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid. For all transfection experiments, we plated the cells in clear 24well tissue culture plates between 312. Suspension cells can be plated the day of transfection. I want to try to use hela cell and lipofectamine 2000 to.

For adherent cells, we recommend 9095% confluence at the time of transfection for high efficiency, high expression levels, and to minimize cytotoxicity. Transfection protocol transfection components component cat. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine 2000 5ul, 10ul in the 6well plate. Protocols for transfecting the most common cell lines with. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. A specific, precalculated amount of the reagent needs to be complexed with dnarna in optimem medium. Mar 27, 2007 according to the manufacturer, and my own experience using human embryonic kidney cells, hek 293, lipofectamine 2000 fulfills both of these criteria. Protocol 2 producing lentivirus in hek293t cells using a 2. Hek293 cells, bj fibroblasts, and ecs were transfected with 1.

Lipofectamine 3000 is a new transfection reagent which is claimed to be more efficient than other transfection reagents like turbofect. Protocol 2 producing lentivirus in hek293t cells using a. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Would anyone have an efficient protocol for transfection. Seed cells onto the wellplates petridishes to be transfected so that day 1 is at 7080%. Similar results were obtained on transfection of hek 293f cells with lipofectamine 2000 invitrogen.

Hek293t human embryonic kidney cell line 293t crl3216, from the atcc cell bank, usa or 293t acc 635 from the dsmz cell bank, germany. I want to try to use hela cell and lipofectamine 2000 to do. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. With automated, highthroughput systems, a complexing volume of 50 l is recommended for transfections in 96well plates. Transfection efficiency and protein expression using geneart crispr nuclease vector. Passage cells every 34 days to ensure that they do not enter senescence. Transfection of human hek 293f cells with metafectene pro. Lipofectamine 2000 cd reagent thermo fisher scientific. For a 12 well plate, i would start with about 12ug dna and 26ul lipofectamine for each well. Then, cells were washed with pbs twice and transduced with scaav2cbapegfp at 2,000 vgs per cell. This protocol describes the steps needed for successful transfection of hek293 cells adapted to serumsupplemented or serumfree medium in suspension culture using linear pei. Stealthsirna transfection protocol lipofectamine 2000.

Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Transfection efficiencies lower than 50% were not reported in this table. Expression in the presence of various transfection reagents a hela cells were treated with different transfection reagents for 6 hr without plasmids, according to the companys manual. Cotransfection protocol for multiple plasmid dnas mirus bio.

The cell culture must have 90% viability and be 85% confluent on the day of transfection. Dna is enclosed within the liposomes and the complexes fuse with the host cell membrane for dna entry into the cytoplasm and eventu ally into the nucleus. Lipofectamine 3000 that displayed strong toxicity to hek293 cells 65. Transfection protocol lipofectamine 3000 transfection. Can anyone provide me with a proper transfection protocol. Lipofectamine messengermax reagent protocol outline a. Cas9 protein and hprt1 grna were transfected into hek293 and hct116 cell lines a. Improved delivery of cas9 proteingrna complexes using. Thermo scientific nunc 24well cell culture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. The examples given within the protocol are for 96well plates, and table 1 provides the transfection reagent volumes for additional plate types. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. In our experience, hek293t cells are easily transfected and the percentage of transfection can be around 80% by using the present protocol. Rapid and highly efficient mammalian cell engineering via.

D after 20 min incubation a add the dna lipofectamine mix to the cells 1 mla 5 ml, the total medium will be 6 ml. Prewarm 50ml of optimum stored in cold room at 4c 10min. Plate cells for transfection only 1 day before the experiment. Materials needed have the following reagents on hand before beginning. A preoptimized hek293 transfection kit is available from altogen biosystems, which includes. They are also known, more informally, as hek cells. Transfecting a human neuroblastoma cell line with monster. In this study, the optimal dose of lipofectamine 3000 and turbofect to transfect adherent cell lines such as chok1 and hek293 cells in comparison with nonadherent h9t cells with pegfpn1 and pcdh was identified. In polypropylene microfuge tubes do not use polystyrene tubes, make a cocktail for each transfection.

Add 6l of lipofectamine2000 from biostores and 100 l of. Hek293 is a cell line derived from human embryonic kidney cells grown in tissue culture. We have found that transfection is especially challenging when mouse embryonic stem mes cells are cultured in the newly. Comparison of transfection efficiency of polymerbased and. Membrane fusion inducers, chloroquine and spermidine. Jun 30, 2016 reliable and efficient dna and rna transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. Would anyone have any efficient protocol for transfection of the pcdna3. Transfection of hek293ebna1 cells in suspension with linear pei for production of recombinant proteins csh protoc. The transfection efficacy of lipofectamine 2000 was compromised by. Transfection of cells should be performed only between passages 5 and 25 postthaw. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Protocols for transfecting the most common cell lines with x. Check gfp expression around 24 hours after transfections.

Lipofectamine and nucleofection of hek 293 were essentially equivalent in terms of toxicity and efficiency. In this study, transfection efficiency and volumetric productivity following transient gene expression in hek293 cells were evaluated using green fluorescent protein gfp and human placental secreted alkaline phosphatase seap as reporter genes. This protocol is based on that from life technologies called. Dharmafect transfection reagentssirna transfection protocol. The following is a general protocol for use of dharmafect transfection reagents to deliver sirna into cultured mammalian cells.

Lipofectamine 2000 is a cationlipid formulation transfection reagent that forms liposomes in aqueous media. Transfection was investigated using various reagents, transfast, transfectam, lipofectamine 2000, and transitneural reagents, and initial experiments demonstrated. Silencing of gene expression in cultured cells using small. If you let them reach confluency, even if this happened a few passages ago, the transfection efficiency will be. Optimizing transfection lipofectamine 2000 cd reagent.

For the hardtotransfect cell line such as hepg2, we recommend the reverse transfection. Transfection guide overview of transfection methods promega. A table listing recommended blockit fluorescent oligo and lipofectamine 2000 amounts and volumes to use for transfection in different tissue culture formats. Cell transfection in a 24well plate using crisprmax. Higher densities can lead to spontaneous differentiation. Maintain p19 cells undifferentiated in mem with 10% serum 7. I am currently having problems with my transfections and request help.