Optimized conditions for successful transfection of human. Cells in a 6 well dish, plated and at 70% confluence. Comparative expression of gfp in h9c2 rat cardiomyocytes transfected with viromer red and lipofectamine 2000 fig. Lipofectamine 2000 reagent thermo fisher scientific. Preparation of cells in suspension the following protocol is given for transfecting mcf10a derivatives in 6well plates.
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. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. All these cells were transfected by lipofection, using the lipofectamine 2000 reagent. Lipofectamine transfections once working now dying cell. Comparative transfection of dna into primary and transformed. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Transfection was measured by luciferase activity using a conventional assay.
The animal protocol was approved by the subcommittee on research animal care in. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. For this study, we used a human mammary epithelial cell line mcf10a, which is. Transfex transfection of plasmid dna into mcf7 cells. Hek293t cells were transfected with lipofectamine 2000 invitrogen in accordance with the manufacturers instructions. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. After 2 years of working with mcf10a cells i came to the conclusion that pei works the best. Cells should be plated on collagencoated dishes, as for normal propagation. Genjet in vitro dna transfection reagent for mcf10a cell ver. In my experience lipofectamine works like a charm in many cells, including hek293. Lipofectamine 2000 transfection reagent dartmouth college. Has anyone used lipofectamine 3000 for plasmid transfection.
If you find this doesnt work for your specific cell type, then you can you look to our cell specific protocols for further optimization. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. For all transfection of multiple wells in 6 well plate, multiply cdna, optimem, and lipofectamine quantity used by number of wells to transfect. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Dna complexes were added to wells so that transfection could occur as cells were attaching to the plate. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. At the moment we are using lipofectamine ltx plus, but the transfection efficiency is low. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. Characterization of the estrogen receptor transfected. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Plate cells for transfection only 1 day before the experiment. Mcf10a cells should be plated in a 10 cm plate 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal 95100% confluency at the time of transfection. 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.
Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Lipofectamine 2000 lf was used as transfection agent, and. The day before transfection, dissociate cells that are 8090% confluent in a t75 flask. The vector contained an ofp reporter gene and was transfected. Lipofectamine transfection of c2c12 cells bridges lab. Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectamine 2000 and lipofectamine rnaimax transfection. Genscript recommends using lipofectamine 2000 for all transfections.
At the moment we are using lipofectamine ltx plus, but the transfection efficiency. 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. Our lipofectamine reagent protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Invitrogen lipofectamine 2000 transfection reagent. Atcc achieved transfection efficiencies of 8590%, using the protocol described below. Nov 20, 2008 in my experience lipofectamine works like a charm in many cells, including hek293. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent. We are grateful to steve santer, robert pauley, and fred miller for providing the mcf10a cells and for instructions on their growth. Gibco hams f12 nutrient mix 11765054 5% horse serum 1 mm gibco sodium pyruvate 160070 25 mm gibco hepes 15630106 proper culture techniques and procedures are an essential. 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. Human mcf10a mammary epithelial cells undergo apoptosis. Jul 29, 2009 i have been transfecting hek293 cells successfully with a gfp fusion protein using lipofectamine 2000. Highest transfection efficiency in many cell types and formats e. The highest transfection efficiency in many cell types and formats e.
Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. In tube a place 2 g of dna in optimemi for a final volume of 200 l. The transfection efficiency is still low, but its better than all the other transfection reagents and. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the.
Lipofectamine transfections once working now dying optimised transfection in heks starting to cause cell death jul292009 hi, i would be very grateful if anyone can suggest anything to help me here. Count the cells using standard trypan blue exclusion. Preparation of cells in suspension the following protocol is given for transfecting mcf10a derivatives in 6. Refer to the cell lines database for a list of cell types successfully. To transfect cells with sirna, follow the protocol as described for dna but. This is often the best place to start, especially in a new cell line. Transfection protocol lipofectamine 3000 transfection. Invitrogen lipofectamine 3000 transfection reagent fisher. Lipofectamine 2000 and lipofectamine rnaimax transfection reagents introduction life technologies is committed to providing researchers and scientists the best products, enabling cutting edge technology while keeping the environment in mind. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Mcf10a and sum cell lines were cultured in a special dmemf12 medium.
It is best to use polypropylene instead of polystyrene tubes. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. I use 24well plates for transfections in triplicates. Lipofectamine transfections once working now dying cell biology. Supplemental data supplemental experimental procedures cell.
Dharmafect volumes per well represent guidelines and need to be optimized. In biontex experiments gfp positivity increased to 90. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Populations of mcf10a cells that were marked by gfp transfection and scored for lainduced bax localization showed that disruption of the actin cytoskeleton led to an approximately 10fold increase in the number of cells displaying mitochondrial bax localization fig. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. Mcf10a cells were reverse transfected in 384well plates 1,000 cells per well. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. I have been transfecting hek293 cells successfully with a gfp fusion protein using lipofectamine 2000. It is recommended that cells are growing at 9095% confluence prior to transfection for adherent cells. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate.
For additional information on scaling your transfection reaction, see page 4. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Transient plasmid dna transfection into hela and hacat. To investigate the influence of medium and transfection time, primary hepatocytes were transfected with metafecte pro, lipofectamine 2000 or targefect in the presence of dmem figure 2a or optimem, withwithout virofect enhancer, for 3 hours or 16 hours figure 2a, 2b, 2c.
Comparative nucleic acid transfection efficacy in primary. Dna complexes were added to cells that had already been allowed to attach to the plate b. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Transfecting plasmid dna into mcf10a cells using lipofectamine. Mcf10a cells grown in complete medium on plastic slides were transfected with 10. Posttranscriptional regulation of the gasc1 oncogene with active. Advanced transfection with lipofectamine 2000 reagent. The lenti293t cell line has been tested as a suitable cell line for lentiviral production due to high transfection efficiency, high level of protein yield and high titer of lentiviral production.
Luciferase activity was measured after 24 hours figures 24. I am looking for a protocol for plasmid transfection into mcf10a cells. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. The presence of optimem resulted in significantly higher transfection rates when lipofectamine 2000 was used.
I agree with parvoman that one of the main considerations is how well the cells are growing. A sample protocol is listed here for transfection experiments performed in 6well plates. Help on transfecting mcf7 and hct 116 cells transfection. Lipofectamine 2000 transfection reagent from invitrogen. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection.
Transfection protocol lipofectamine 3000 transfection reagent. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. This protocol is for transfecting plasmid dna into mammalian cells. Htb22 is an epithelial, breast adenocarcinoma cell line that was derived from a metastatic pleural effusion. Lipofectamine 2000 transfection protocol 10cm plates reagents. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Invitrogen lipofectamine 3000 transfection reagent. After drug or dmso treatment, each plate was median centered to 2,000 cellswell to. Mcf10a cells were cultured as described debnath et al. Transient plasmid dna transfection into hela and hacat cells.
Using lipofectaminez 2000 for transfection provides the following advantages. If designing an experiment that involves transfection, ensure that setup coincides with a cell passage. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Transfection protocols thermo fisher scientific tr. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. Characterization of the estrogen receptor transfected mcf10a.
Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. I optimised the transfection as suggested by the manufacturer and found them to be successful at all ranges tested lipofectamine. The media needs to be changed at least 1hr prior to transfection. Cells were transfected with a firefly luciferase encoding plasmid dna 0. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. If you let them reach confluency, even if this happened a few passages ago, the transfection efficiency will be significantly diminished. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Add 6l of lipofectamine2000 from biostores and 100 l of. Transfecting plasmid dna into mcf10a cells using lipofectamine 3000 reagent. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. The nucleofection resulted in a transfection efficiency of 90% and cytotoxicity of 5%.
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