Invitrogen lipofectamine 2000 transfection reagent 15ml. It is best to use polypropylene instead of polystyrene tubes. Highest transfection efficiency in many cell types and formats e. In this study, lipofectamine reagent and a plasmid with a universal cmv promoter were preferred owing to a relatively high efficiency reported with leukaemia cells in previous studies uchida et al.
Transfection of myeloid leukaemia cell lines is distinctively. For additional information on scaling your transfection reaction, see page 4. Superior performance for cotransfection of sirna and plasmid dna. Lipofectamine 2000 reagent thermo fisher scientific. Enhanced mrna delivery into lymphocytes enabled by lipid. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Lipofection of the human t lymphoblastic leukaemia tall. Transfection efficiency % 80 100 60 40 20 0 thp1 jurkat primary mes fibroblasts jetmessengermrna lipofectamine 2000dna indel % 30 60 20 50 10 40 0 a549 hek293 jetcrispr lipofectamine crisprmax request your sample of jetprime, jetcrispr, jetmessenger or interferin on. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to deliver invitrogen.
Jurkat transfection by electroporation harvard university. Smallsized, stable lipid nanoparticle for the efficient. Unique formulation maximize transfection performance in jurkat cells and other hardtotransfect cells such as k562, raw 264. I also tried the transitjurkat transfection reagent from mirus. Transfection of jurkat cells use this procedure to transfect plasmid dna into jurkat cells in a 24well format for other formats, see scaling up or down transfections, below. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. High efficiency delivery achieve 510% transfection efficiency in jurkat cells to ensure experimental success. The commonlyused plasmid dna or rna lipofectamine transfection reagent had been shown to be able to deliver many proteins into cells, so we tested several lipofectamine variants for their ability to efficiently deliver cas9 rnps sells et al. Jurkat, human tcell leukemia, plasmid dna, lipofectamine 3000, l3000008 l3000015 pdf.
Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Transfection of cultured eukaryotic cells using cationic lipid. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. All amounts and volumes are given on a per well basis. Rnaimediated knockdown of vdr surprisingly suppresses. Cellspecific transfection protocols thermo fisher scientific us. 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. Invitrogen lipofectamine 2000 transfection reagent fisher. Invitrogen lipofectamine transfection reagent protocols have been.
Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Apr 10, 2012 in contrast, exposure of cells to the nanocomplexes with polybrene pretreatment achieved significantly high cell transfection and gene expression rates. Experimental procedures transfection protocol one day before transfection jurkat cells were plated in 1 ml of rpmi 1640 medium supplemented with 10% fcs, 1% hepes and lglutamine 2mm in a 12well culture plate at the concentration of 100. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Jurkat cells human tcell lymphomaleukemia and k562 human. We have tried many transfection methods in tcells, but none of them have given us adequate results. Transfection and genome engineering fisher scientific.
Jurkat transfection by electroporation odified, j exp med. Transfection efficiency and protein expression using geneart crispr nuclease vector. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. After transfection for 48 hr, gfpexpressing cells were quantified by flow cytometry analysis. In contrast to the efficient transfection in the rapidly dividing jurkat cells, the transfection into nondividing human islets was low and required prolonged incubation. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Viafect transfection reagent transfection assay e4981. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Mammalian cell penetration, sirna transfection, and dna. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format.
The optimal amount of dna to use in the transfection will vary widely depending upon the type of dna, transfection reagentmethod, target cell line, and number of cells genetic material dna quality and quantity factors affecting transfection 8. Lipofection of the human t lymphoblastic leukaemia tall cell line jurkat. Vpu were mixed with actinrfp expressing jurkat targets and imaged immediately. Scaling up or down lipofectamine 3000 reagent transfections. Cells in a 6 well dish, plated and at 70% confluence. Jurkat cells shouldnt be that difficult to transfect. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Transient plasmid dna transfection into hela and hacat cells. In tube a place 2 g of dna in optimemi for a final volume of 200 l. In contrast, exposure of cells to the nanocomplexes with polybrene pretreatment achieved significantly high cell transfection and gene expression rates.
Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. 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. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Rapid and highly efficient mammalian cell engineering via cas9 protein transfection. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Caco2 transfection reagent colon carcinoma cells altogen. One day before transfection jurkat cells were plated in 1 ml of rpmi 1640 medium supplemented with.
This shows that the lipofectamine 2000 concentration should be optimized for each cell type. Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco2 colon cancer cell line. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Rnaimediated knockdown of vdr surprisingly suppresses cell. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Smallsized, stable lipid nanoparticle for the efficient delivery of sirna to human immune cell lines. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. A sample protocol is listed here for transfection experiments performed in 6well plates. This reference provides a recommended procedure to transfect plasmid dna into human jurkat human tcell leukemia cells atcc cat. However, transfection rates of lipofectamine with plasmid dnas in the. Followingthe transfection, the cells emitted green light under. Lipofectaminepdna and superfectpdna complexes showed high transfection efficiency in rapidly dividing jurkat cells, but low transfection in nondividing human islets. If using lipofectamine 2000, fresh transfection medium is not required.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Optimized conditions for successful transfection of human. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Research article rnaimediated knockdown of vdr surprisingly suppresses cell growth in jurkat t and u87mg cells zeinab shirvanifarsania, mehrdad behmaneshb, a department of cellular and molecular biology, faculty of life sciences and technology, shahid beheshti university g.
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. Invitrogen lipofectamine 2000 transfection reagent. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. Based on our experiences, the lipofectaminemedicated transfection routinely resulted in gfp gene expression in 24 % with or without polybrene pretreatment of the tested human lymphomaleukemia cells. Wash the cells 2x pbs seed 10 6 cells in 300 ul warm optimem in 24 wellsplates. Lipofectamine transfection of c2c12 cells bridges lab. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. To simplify the transfection optimization process, we have created a. 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. Rapid and highly efficient mammalian cell engineering via. Dec 06, 2009 in this study, lipofectamine reagent and a plasmid with a universal cmv promoter were preferred owing to a relatively high efficiency reported with leukaemia cells in previous studies uchida et al. Lipofectamine 3000 reagent transfection efficiency.
Higher densities can lead to spontaneous differentiation. I use 24well plates for transfections in triplicates. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. The intracellular trafficking mechanism of lipofectamine. Genscript recommends using lipofectamine 2000 for all transfections. Following 48 h posttransfection, only a small percentage of hl60 cells cultured in suspension were positive for egfp. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start.
Transitjurkat transfection reagent research reagent. The day of transfection, count the cells to determine culture density. Transitjurkat transfection reagent research reagent products. It was very efficient, but all the transfected cells died. Precipitate 5 pmol around 10 g plasmid dna in 100 l. Lipofectamine 3000 and rnaimax outperformed lipofectamine 2000 in hek 293 cells figure s1.
Transitjurkat transfection reagent is specifically optimized to provide exceptional transfection efficiency of plasmid dna in jurkat cells and cell types of. Cationic lipid and polymerbased gene delivery to human pancreatic islets. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Each cell line was transfected with a luciferase reporter plasmid using either viafect transfection reagent, lipofectamine 2000 transfection reagent or lipofectamine 3000 transfection reagent following manufacturer instructions, applying either 5l 50ng of dna or 10l 100ng of dna of the transfection complex in each well of a 96. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. In tube b place 190 l of optimemi and 10 l lipofectamine 2000.
Using lipofectaminez 2000 for transfection provides the following advantages. Lipofectamine 2000 transfection reagent dartmouth college. Transfecting the hardtotransfect lymphomaleukemia cells. Maintain p19 cells undifferentiated in mem with 10% serum 7. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Each tube gets 8mls of optimem, 32ug of gag pol, 12. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Whats your transfection protocol for lipofectamine. Dharmacon rna technologies, lipofectamine 2000 invitrogen. 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. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. The transfection protocol for lipofectamine 3000 reagent was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. What is the best method to transfect tcells jurkat. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3.
Prewarm 50ml of optimum stored in cold room at 4c 10min. It is possible to plate and transfect the cells on the same day, but cells must adhere to. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. High transfection efficiency of jurkat cells with neon tranfection system. Add 6l of lipofectamine2000 from biostores and 100 l of. The highest transfection efficiency in many cell types and formats e. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Despite this, a satisfactory mechanismbased explanation of their.
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