Hi, i have a question about the use of lipofectamine 2000 for transfection of 293t cells. Altogether, the silencing with lipofectamine resulted in 400% of silenced cells. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Ag n and 1, 5, 10 or 20 l of lipofectamine were dissolved, each in 50 l of optimem i reduced serum medium without phenol red invitrogen. Transfection protocol lipofectamine 3000 transfection reagent hepg2 liver cancer cells complete growth medium component cat. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from a difficult to transfect cell line that had been giving us trouble with the lipofectamine 2000. The helaegfp cells were dividing about two times faster than the hela cells. A rna was harvested from hela cells that were transfected with a non.
For each transfection sample, prepare dnalipofectamine 2000 complexes as follows. Padma nambisan, in an introduction to ethical, safety and intellectual property rights issues in biotechnology, 2017. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng. Livecell imaging to compare the transfection and gene. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. George gey in the colored ward of the johns hopkins hospital. Transfection protocol lipofectamine 3000 transfection. I want to try to use hela cell and lipofectamine 2000 to do transfection. Lipofectamine transfection of c2c12 cells bridges lab.
Efficiently transfect difficulttotransfect cells lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common cells e. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Lipofectamine 2000 reagent thermo fisher scientific. 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. Aspirate the medium from the dish of cells, add the diluted transformation mix, and incubate the cells with the mix in a 10% co 2 incubator for approximately 5 h. Cells in a 6 well dish, plated and at 70% confluence. I want to try to use hela cell and lipofectamine 2000 to. 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. The trusted, simple, broadspectrum transfection reagent for most cell lines. I use 24well plates for transfections in triplicates.
Some of my cells will dried if i used the volume suggested by the manual. Highest transfection efficiency in many cell types and formats e. Transfection efficiency and protein expression using geneart crispr nuclease vector. Lipofectamine 3000 transfection reagent hepg2 liver. Arpe19 transfection kit retinal pigment epithelium. 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. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.
Rapid isolation of transfectiongrade plasmid dna in high yields. 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. Invitrogen lipofectamine 3000 transfection reagent our top recommended reagent for dna delivery with the power to transfect your most difficult cells superior performance10fold higher transfection efficiency into difficult cells versatileenables the highest transfection efficiency in. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates.
Transfection efficiency comparison in hela cells using transfectamine. The processing of dna for transfection and short interfering rna sirna. Gibco rpmi 1640 medium with glutamax supplement 61870036 10% gibco fbs a3160401 25 mm gibco hepes 1 m 15630080 1. 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. Transfection protocol lipofectamine 3000 transfection reagent. To transfect cells with sirna, follow the protocol as. Mar 09, 2017 the processing of dna for transfection and short interfering rna sirna.
Add 6l of lipofectamine2000 from biostores and 100 l of. Transfection of living hela cells with fluorescent poly. I do have cell toxicity, but not so serious as yours. Altogen biosystems offers the hela transfection reagent kit for the hela cervical cancer cell line, an invaluable tool in conducting a broad array of. Hela transfection kit cervix adenocarcinoma altogen. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Also, hela cells were used by jonas salk to test the first polio vaccine in the 1950s. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000.
A hela cells were transfected with 100 nm of sirna duplexes targeting jnk1 and parp1 cell signaling technology using lipofectamine 2000 and optimem i invitrogen by a reverse transfection. Transfection protocol lipofectamine 3000 transfection reagent ncih1299 lung cancer cells complete growth medium component cat. Versatileone reagent for dna, rna, and cotransfection. Transient plasmid dna transfection into hela and hacat. We did not find substantial transfection efficiency variations in. Twentyfour hours posttransfection the cells were harvested and cell viability assayed. Comparative transfection efficiency of jetprime versus its main competitor. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. In the latter case, hela cells that stably expressed egfp were used. General procedure for transfection of mammalian cells with. Transfection protocol lipofectamine 3000 transfection reagent lncap prostate cancer cells complete growth medium component cat. Invitrogen lipofectamine 3000 transfection reagent. Lipofectamine tm 2000 invitrogen was used to aid the transfection of hela cells with the c 24. In biontex experiments gfp positivity increased to 90.
I want to try to use hela cell and lipofectamine 2000 to do. 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. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. What i found is lipofectamine 2000 is toxic to cells, so i reduced the recommended quantity of the invitrogen protocol. Plasmid transfection influences the readout of nonsense. Improved cell viabilitygentle on your cells, with low toxicity. Transient plasmid dna transfection into hela and hacat cells. For comparison, the commercial liposomal transfection agent lipofectamine was used. For hela cells we have already achieved a transfection efficiency of 87, 8 97,8 % with lipofectamine. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays.
Genetic transfection an overview sciencedirect topics. Maintain p19 cells undifferentiated in mem with 10% serum 7. The mechanism of lipofectamine 2000 mediated transmembrane. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Lower confluence will achieve higher percentage of transfection but may yield a lower absolute number of transfected cells. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul. This allows the dna and the lipofectamine to form the complexes that will get the dna into the cells. In hela cells subjected to lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437.
Transient transfection of mammalian cells with lipofectamine. They have also been used in aids research, effects of radiation and toxic substances and gene mapping. Gibco rpmi 1640 medium, with glutamax supplement 61870036 10% gibco fbs a3160401 1 mm gibco sodium pyruvate 160070 proper culture techniques and procedures are an essential part of ensuring successful. Gently mix media on cells via figure 8 pattern and back and forth motions. Plate cells so they will be 7090% confluent at the time of transfection. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Prepare plasmid dnalipid complexes recommend 2 doses of lipid.
Luciferase expression rlu and cell viability % in hela, cho and hek standard cell lines after pcmvluc plasmid transfection with viromer red, viromer yellow and 4 leading competitors benchmark analysis lipocalyx gmbh vr01lb00, vr01lb01 hela. Increasing concentrations of plasmid dna encoding for human fbl pcmv2fbl were transfected into hela cells using lipofectamine 2000. The amount of lipofectamine 2000 should be changed in proportion to the culture medium. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000. Gibco dmem with glutamax supplement 10566016 10% gibco fbs a3160401 proper culture techniques and procedures are an essential part of ensuring successful transfection. 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. Lipofectamine 2000 dna transfection reagent protocol. Hela cells at 50% and 90% confluency were transfected in duplicate using 3 l of transitlt1 reagent or 4 l lipofectamine 2000 lf2k per well of a 12well plate. L3000001 lipofectamine 3000 transfection reagent sparshbio. Total rna levels were decreased in samples transfected with lipofectamine 2000 for 24 hours.
Transfection and genome engineering fisher scientific. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Hela to, 293 msr and u2os cells were transfected using calcium phosphate transfection and mcf7 using jetprime transfection reagent. But unfortunately the survival rate of cells has only been 46,9 61,9 %. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. Prewarm 50ml of optimum stored in cold room at 4c 10min.
With automated, highthroughput systems, a complexing volume of 50 l is recommended for transfections in 96well plates. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. Gfp fluorescence was determined by facs analysis see figure 1. The hela cell line was developed in the 1950s from a particularly aggressive strain of cervical cancer cells taken during a routine biopsy from a 30yearold mother of five, henrietta lacks. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. I am doing transfection using lipofectamine 2000 and plasmid dna, too.
Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Refer to the cell lines database for a list of cell types successfully. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Invitrogen lipofectamine 3000 transfection reagent our top recommended reagent for dna delivery with the power to transfect your most difficult cells superior performance10fold higher transfection efficiency into difficult cells versatileenables the highest transfection efficiency in the broadest range of cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. You can not use antibiotics during transfection step because it will cause cell death. Successfully transfect the widest variety of biologically relevant cell types with a reagent that offers. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Lipofectamine 2000 transfection reagent dartmouth college. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium.
On the day of transfection just prior to preparing complexes, plate 1. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Original article an improved method for increasing the efficiency of. Higher densities can lead to spontaneous differentiation.