Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetpei and expressfect. On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using lipofectamine 3000 transfection reagent. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Transfecting plasmid dna into nih3t3 cells using lipofectamine ltx reagent. Transfection protocol lipofectamine 3000 transfection reagent.
Invitrogen has taken the time to optimize this reagent and the recommend protocol that they provide. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual. Tib202 is a human acute monocytic leukemia cell line that was derived from peripheral blood. This reference provides a recommended procedure to transfect plasmid dna into thp1 human monocytic leukemia cells atcc cat. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. I want to try to use hela cell and lipofectamine 2000 to. Transient plasmid dna transfection into hela and hacat cells. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437.
Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Prewarm 50ml of optimum stored in cold room at 4c 10min. In tube a place 2 g of dna in optimemi for a final volume of 200 l. The highest transfection efficiency in many cell types and formats e.
Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Highly efficient transfection of human thp1 macrophages. Genscript recommends using lipofectamine 2000 for all transfections. Maximal transfection efficiency in mc3t3e1, c3h10t12, hela, c2c12, hep g2, and hct116 was seen using fugene hd, in pt30, 4t1, and hekn was seen using arrestin, and in mcf7 was seen using jetpei. Dna complexes were added to cells that had already been allowed to. Aug 17, 2017 the pcmvmyc3mdm2 plasmid or small interfering rna was transfected into cells using oligofectamine invitrogen, 12252011 or lipofectamine 2000 invitrogen, 11668019 according to the. Lipofectamine 2000 transfection reagent from invitrogen. Genexplus transfection of plasmid dna into thp1cells. 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. Lipofectamine 2000 reagent thermo 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. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity.
I am doing crisprcas9 transfection in sw620 and ht29 cell lines using lipo2000. In biontex experiments gfp positivity increased to 90. Invitrogen lipofectamine 3000 transfection reagent 0. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization.
I use 24well plates for transfections in triplicates. 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. Xtremegene hp reagent xhp, and competitor reagents ltx and l2k. Comparative transfection efficiency of jetprime versus its main competitor.
I have successfully transfected hct116 cells, but have been told ht29s are more difficult to transfect. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Optimized conditions for successful transfection of human. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. The media needs to be changed at least 1hr prior to transfection. Enable high transfection efficiency in novel genome editing applications. Ht29 transfection kit colorectal adenocarcinoma altogen. Lipofectamine 2000 dna transfection reagent protocol. Hi everyone, can anyone suggest a protocol for transfection of ht29 cells with sirna using lipofectamine 2000. The mixture is used to transfect hek293 cells in a. While lipofectamine 2000 reagent demonstrated transfection efficiencies. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. B hela cells were transfected with luciferase encoding plasmid dna using either transit.
A highly efficient method of transfecting hep g2 cells. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Highest transfection efficiency in many cell types and formats e. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Transfection efficiency of p19 cells is between 3050%. Lipofectamine 2000 transfection reagent dartmouth college. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Jul 24, 2007 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. Transfecting plasmid dna into ht29 cells using lipofectamine. Choosing the right transfection reagent for optimal efficiency.
Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Invitrogen lipofectamine 3000 transfection reagent l3000008. Using lipofectaminez 2000 for transfection provides the following advantages. I have got transfection efficiency of 3040% in sw620 and less than 30% in ht29.
The transfection efficiencies of the pte cells were 5%. I want to try to use hela cell and lipofectamine 2000 to do. The transfection efficiencies of the pte cells were 5% effectene. Transient plasmid dna transfection into hela and hacat. Fugene hd, jetpei, lipofectamine 2000, and superfect for gene transfection. Xtremegene sirna transfection reagent is a proprietary blend of lipids and other components, free of animal products. Transfection was measured by luciferase activity using a conventional assay. In another hand, i have tried the same lipofectamine under the given experiemental conditions to ht29 cells. Transfecting plasmid dna into nih3t3 cells using lipofectamine. Transfection protocol of lipofectamine rnaimax can be. Sep192012 hi everyone, can anyone suggest a protocol for transfection of ht29 cells with sirna using lipofectamine 2000. Dna complexes were added to cells that had already been allowed to attach to the plate b.
Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. A sample protocol is listed here for transfection experiments performed in 6well plates. Cells were transfected with a firefly luciferase encoding plasmid dna 0. Lipofectamine transfection of c2c12 cells bridges lab. This protocol presents an efficient and reliable method to transfect human thp1 macrophages with sirna or plasmid dna by electroporation with high transfection efficiency while. Transfection protocol lipofectamine 3000 transfection. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent. For additional information on scaling your transfection reaction, see page 4. It is used to increase the transfection efficiency of rna. Transient plasmid dna transfection protocol for ht29 cells per well of a 96well plate using xtremegene hp dna transfection reagent. Transfection protocol lipofectamine 3000 transfection reagent ncih292 lung cancer cells complete growth medium component cat. It is best to use polypropylene instead of polystyrene tubes. Transfection of ht29 cells with sirna and lipofectamine 2000. Advanced transfection with lipofectamine 2000 reagent.
Can anybody recommend a method transfection substrate of how to transfect ht29 cells. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using lipofectamine 3000 transfection. Lipofectamine is formulated to give high transfection efficiency with little. Transfection guide overview of transfection methods promega. 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 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. The nucleofection resulted in a transfection efficiency of 90% and cytotoxicity of 5%. Using four different transfection reagents fugene hd, lipofectamine 2000, effectene, and lipofectamine ltx with plus reagent, jacobsen et al. Protocol for ht29 sirna transfection using lipofectamine. Lipofectamine messengermax and the leading mrna delivery reagent were used to deliver gfp mrna 500 ngwell in a 24well format. Xtremegene transfection reagents general recommendations.
Invitrogen lipofectamine 2000 transfection reagent. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. This reference provides a recommended procedure to transfect plasmid dna into ht29, human colon adenocarcinoma cells atcc no. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. The vector contained an ofp reporter gene and was transfected. To transfect cells with sirna, follow the protocol as described for dna but. Invitrogen lipofectamine 3000 transfection reagent fisher. The pcmvmyc3mdm2 plasmid or small interfering rna was transfected into cells using oligofectamine invitrogen, 12252011 or lipofectamine 2000 invitrogen, 11668019 according to. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for.
Cells in a 6 well dish, plated and at 70% confluence. Explore our xtremegene transfection reagent recommendations guide for the. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Add 6l of lipofectamine2000 from biostores and 100 l of. It is recommended that cells are growing at 9095% confluence prior to transfection for adherent cells. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Comparative transfection of dna into primary and transformed. Comparison of transfection efficiency of nonviral gene. Invitrogen lipofectamine 3000 transfection reagent.
Refer to the cell lines database for a list of cell types successfully. Comparison of transfection efficiency of nonviral gene transfer reagents. Invitrogen lipofectamine 2000 transfection reagent fisher. This reference provides a recommended procedure to. Help on transfecting mcf7 and hct 116 cells transfection. In a majority of cases, one wants the highest level of transfection. Xtremegene sirna transfection reagent polymer reagent for. Can anyone suggest a protocol for transfection of ht29 cells with sirna using lipofectamine 2000. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Xtremegene sirna transfection reagent polymer reagent. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Lipofectamine messengermax outperforms leading dna delivery reagent and leading mrna delivery reagent in various cell lines that are very difficult to transfect bend. 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.
In tube b place 190 l of optimemi and 10 l lipofectamine 2000. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. To perform transfection experiments in other cell culture plates, simply multiply the. Gibco rpmi 1640 medium with glutamax supplement 61870036 10% gibco fbs a3160401 25 mm gibco hepes 1 m 15630080 1. In my experience lipofectamine works like a charm in many cells, including hek293. If there is anyone who have tried best protocol to trasfect ht29 bothe reagent and condition, it will be really great to know. 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. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. The mechanism of lipofectamine 2000 mediated transmembrane. Improved delivery of cas9 proteingrna complexes using.
I have used lf2000 successfully with the following cell lines. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Mdm2 promotes epithelialmesenchymal transition and. I agree with parvoman that one of the main considerations is how well the cells are growing. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. The fugene hd protocol database provides optimization guidelines for a. Dna complexes were added to wells so that transfection could occur as cells were attaching to the plate. Protocol for ht29 sirna transfection using lipofectamine 2000. However, significant toxicity from lipofectamine 2000 was also.