It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Jurkat cells human tcell lymphomaleukemia and k562 human. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Whats your transfection protocol for lipofectamine.
Caco2 transfection reagent colon carcinoma cells altogen. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to deliver invitrogen. If using lipofectamine 2000, fresh transfection medium is not required. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Highest transfection efficiency in many cell types and formats e. I also tried the transitjurkat transfection reagent from mirus. Transitjurkat transfection reagent is specifically optimized to provide exceptional transfection efficiency of plasmid dna in jurkat cells and cell types of.
Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Using lipofectaminez 2000 for transfection provides the following advantages. This shows that the lipofectamine 2000 concentration should be optimized for each cell type. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 2000 reagent thermo fisher scientific. 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. It is best to use polypropylene instead of polystyrene tubes. Invitrogen lipofectamine 2000 transfection reagent fisher.
Viafect transfection reagent transfection assay e4981. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Prewarm 50ml of optimum stored in cold room at 4c 10min. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Invitrogen lipofectamine transfection reagent protocols have been. 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. 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. For additional information on scaling your transfection reaction, see page 4. Jurkat, human tcell leukemia, plasmid dna, lipofectamine 3000, l3000008 l3000015 pdf.
Optimized conditions for successful transfection of human. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Transitjurkat transfection reagent research reagent. Maintain p19 cells undifferentiated in mem with 10% serum 7. Following 48 h posttransfection, only a small percentage of hl60 cells cultured in suspension were positive for egfp.
Transfection and genome engineering fisher scientific. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. Transfecting the hardtotransfect lymphomaleukemia cells using a. Jurkat transfection by electroporation odified, j exp med. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.
High efficiency delivery achieve 510% transfection efficiency in jurkat cells to ensure experimental success. 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. What is the best method to transfect tcells jurkat. 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. Genscript recommends using lipofectamine 2000 for all transfections. 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 3000efficient, reproducible transfection for. Apr 10, 2012 in contrast, exposure of cells to the nanocomplexes with polybrene pretreatment achieved significantly high cell transfection and gene expression rates.
Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. To simplify the transfection optimization process, we have created a. Rapid and highly efficient mammalian cell engineering via. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Lipofectamine transfection of c2c12 cells bridges lab. All amounts and volumes are given on a per well basis.
Lipofection of the human t lymphoblastic leukaemia tall. Vpu were mixed with actinrfp expressing jurkat targets and imaged immediately. Despite this, a satisfactory mechanismbased explanation of their. Jurkat transfection by electroporation harvard university. Smallsized, stable lipid nanoparticle for the efficient delivery of sirna to human immune cell lines. 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.
Transfection efficiency and protein expression using geneart crispr nuclease vector. However, transfection rates of lipofectamine with plasmid dnas in the. Dharmacon rna technologies, lipofectamine 2000 invitrogen. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Wash the cells 2x pbs seed 10 6 cells in 300 ul warm optimem in 24 wellsplates. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Following 48 h post transfection, only a small percentage of hl60 cells cultured in suspension were positive for egfp. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. The highest transfection efficiency in many cell types and formats e. Higher densities can lead to spontaneous differentiation. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. 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. Followingthe transfection, the cells emitted green light under.
Invitrogen lipofectamine 2000 transfection reagent. 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. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. It was very efficient, but all the transfected cells died. Jurkat cells shouldnt be that difficult to transfect. Transient plasmid dna transfection into hela and hacat cells. Jurkat 1 k562 1 l6 8 l929 2 lncap 6 mcf 10a 5 mcf7 2 mdamb231 3 mdamb435 1 mdamb468 9 mdck 1. Lipofection of the human t lymphoblastic leukaemia tall cell line jurkat.
Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. In contrast, exposure of cells to the nanocomplexes with polybrene pretreatment achieved significantly high cell transfection and gene expression rates. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Transfection of myeloid leukaemia cell lines is distinctively.
Smallsized, stable lipid nanoparticle for the efficient. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. This reference provides a recommended procedure to transfect plasmid dna into human jurkat human tcell leukemia cells atcc cat. Invitrogen lipofectamine 2000 transfection reagent 15ml. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
I use 24well plates for transfections in triplicates. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Transient plasmid dna transfection into hela and hacat. 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. Cells in a 6 well dish, plated and at 70% confluence. One day before transfection jurkat cells were plated in 1 ml of rpmi 1640 medium supplemented with. In tube a place 2 g of dna in optimemi for a final volume of 200 l. The day of transfection, count the cells to determine culture density. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. 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. 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. High transfection efficiency of jurkat cells with neon tranfection system. Lipofectamine 2000 transfection reagent dartmouth college.
Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Lipofectamine 3000 and rnaimax outperformed lipofectamine 2000 in hek 293 cells figure s1. Transfecting the hardtotransfect lymphomaleukemia cells. The intracellular trafficking mechanism of lipofectamine. In contrast to the efficient transfection in the rapidly dividing jurkat cells, the transfection into nondividing human islets was low and required prolonged incubation. After transfection for 48 hr, gfpexpressing cells were quantified by flow cytometry analysis. Add 6l of lipofectamine2000 from biostores and 100 l of. 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. Superior performance for cotransfection of sirna and plasmid dna. Mammalian cell penetration, sirna transfection, and dna.
Transfection of cultured eukaryotic cells using cationic lipid. It is possible to plate and transfect the cells on the same day, but cells must adhere to. Lipofectaminepdna and superfectpdna complexes showed high transfection efficiency in rapidly dividing jurkat cells, but low transfection in nondividing human islets. Rnaimediated knockdown of vdr surprisingly suppresses cell. A polymerbased transfection caco2 kit for colon adenocarcinoma cells yields at least 94% transfection efficiency and is a helpful tool for gene expression studies as well as for assessing cellular functionality. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Each tube gets 8mls of optimem, 32ug of gag pol, 12.
Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco2 colon cancer cell line. Rapid and highly efficient mammalian cell engineering via cas9 protein transfection. Transitjurkat transfection reagent research reagent products. 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.
Cationic lipid and polymerbased gene delivery to human pancreatic islets. We have tried many transfection methods in tcells, but none of them have given us adequate results. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. A sample protocol is listed here for transfection experiments performed in 6well plates. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start.
Lipofectamine 3000 reagent transfection efficiency. Cellspecific transfection protocols thermo fisher scientific us. Unique formulation maximize transfection performance in jurkat cells and other hardtotransfect cells such as k562, raw 264. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. 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. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Rnaimediated knockdown of vdr surprisingly suppresses. Precipitate 5 pmol around 10 g plasmid dna in 100 l. Scaling up or down lipofectamine 3000 reagent transfections. Enhanced mrna delivery into lymphocytes enabled by lipid. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1.
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