Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. However, transfection rates of lipofectamine with plasmid dnas in the. Transfection of cultured eukaryotic cells using cationic lipid. Jurkat transfection by electroporation odified, j exp med. We investigated the impact of cell cycle regulation by fibronectin adherence on the transfection of leukaemia cells. The day of transfection, count the cells to determine culture density. Prewarm 50ml of optimum stored in cold room at 4c 10min. Use 3 l of lipofectamine 2000 and 1l of combimag per g of dna.
Transfecting plasmid dna into jurkat cells using lipofectamine ltx reagent. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Does anyone has suitable transfection agent for jurkat cells. In tube a place 2 g of dna in optimemi for a final volume of 200 l. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Invitrogen lipofectamine 2000 transfection reagent fisher. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Transfecting the hardtotransfect lymphomaleukemia cells. An optimized electroporation protocol applicable to a wide range of cell lines. This reference provides a recommended procedure to transfect plasmid dna into human jurkat human tcell leukemia cells atcc cat. This active endosome escape technology is safe and maximizes transfection efficiency as it is using a natural uptake pathway. Cells in a 6 well dish, plated and at 70% confluence.
Jurkat cells human tcell lymphomaleukemia and k562 human. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Do you have a protocol for transfection of suspension cell lines. One day before transfection split the cells at a density of 0.
Add 6l of lipofectamine2000 from biostores and 100 l of. Highest transfection efficiency in many cell types and formats e. Rapid and highly efficient mammalian cell engineering via. 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. Transfecting the hardtotransfect lymphomaleukemia cells using a. An efficient low cost method for gene transfer to t. How does transfection with lipofectamine 2000 work. I need to transfect plasmid dna into jurkat cells, but it seems that these cells are.
Carefully transfer the transfection mix to the lentix 293t packaging cells. To optimize the condition for transfection of jurkat cells with lipofectamine ltx. Pbmcs were obtained from healthy donors, as described above. To transfect cells with sirna, follow the protocol as described for dna but.
Lipofectamine 2000 reagent thermo fisher scientific. Plate cells so they will be 7090% confluent at the time of transfection. Choosing the right transfection reagent for optimal efficiency. I used lipofactamine 2000 and followed the protocol provided by the supplier. Quick reference protocol, msds and certificate of analysis available at mirusbio. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. A sample protocol is listed here for transfection experiments performed in 6well plates. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Yes, please follow the protocol transfection of suspension cell lines, including jurkat and k562, with sirna in 24well plates in the hiperfect transfection. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Jurkat cells used in cell biology research to study cancer of blood cells leukemia. Htm cells were transfected using lipofectamine with a fluorescent mirna dy547 and analyzed 48 hours after transfection. All amounts and volumes are given on a per well basis. To test the transfection efficiency of the reverse transfection approach, a luciferaseencoding plasmid was reverse transfected into hek293, hep g2 and jurkat cells using fugene 6, fugene hd, transitlt1 and lipofectamine 2000 transfection reagents.
Transfection was measured by luciferase activity using a conventional assay. Precipitate 5 pmol around 10 g plasmid dna in 100 l. As always, use good cell culture practices and wear your personal protective. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Jurkat transfection by electroporation harvard university. Cationic lipidmediated transfection of suspension cells with. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. I have used both lipofectamine 2000 and 3000 and always get the same results. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection.
In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Comparative transfection efficiency of jetprime versus its main competitor. 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 transfection of c2c12 cells bridges lab. Transfecting plasmid dna into jurkat cells using lipofectamine. Genscript recommends using lipofectamine 2000 for all transfections. 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. Luciferase activity was measured after 24 hours figures 24. We have tried many transfection methods in tcells, but none of them have given us adequate results. Apr 10, 2012 for further validation, the cell transfection rate of the nanocomplexes was compared to that mediated by lipofectamine, a widely used and commercially available reagent for cell transfection. The following morning, carefully aspirate the media. Jun 15, 2017 a droplet electroporation system has been successfully demonstrated for transfection of jurkat t cell with much higher transfection efficiency 66% than that of conventional system 11.
The procedure for suspension cells transfection is as simple as follows. Jurkat cells were transfected with lipofectamine ltx using plasmids pires 2egfp and. Troubleshooting for transfection of the crisprcas9 using. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Transitjurkat transfection reagent research reagent products. Transfection efficiencies lower than 50% were not reported in this table. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. The jurkat cell line was established in 1970 from the peripheral blood of a patient with acute t cell leukemia. Invitrogen lipofectamine 2000 transfection reagent life. Add the transfection mix dropwise being careful not to dislodge the cells. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Refer to the cell lines database for a list of cell types successfully.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. For further validation, the cell transfection rate of the nanocomplexes was compared to that mediated by lipofectamine, a widely used and commercially available reagent for cell transfection. Maintain p19 cells undifferentiated in mem with 10% serum 7. 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. Refer to magnetofectamine general protocol for optimization. While lipofectamine 2000 reagent demonstrated transfection efficiencies only slightly lower those of viafect reagent, it was considerably more toxic to hela cells at all tested ratios of reagent to dna. Ive generated a plasmid containing my protein of interest lets call him bob with gateway cloning. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng.
Jurkat cells provide a model to study t cell signaling, expression of chemokine receptors susceptible to viral entry, and the effects of radiation. Transfection of jurkat t cells by droplet electroporation. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Higher densities can lead to spontaneous differentiation.
Dec 06, 2009 cell cycle dependence of transfection in fibronectinadherent leukaemia cells. Incubate the cells for 18 h, or until the following morning. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. 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. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Optimization of condition for transfection of jurkat cells. Table 2 examines transfection efficiency and cell viability under optimal conditions for each reagent 24 hours after transfection.
Transfection of myeloid leukaemia cell lines is distinctively. Cultured suspension cells including jurkat, k562 a human myeloid leukemia cell line, and karpas 299 a human anaplastic large cell lymphoma cell line. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Further, we found that the offtarget cleavage rate is reduced using cas9 protein when compared to plasmid dna transfection. In a reverse transfection protocol, cells are added directly to a plate. The protocol involves mixing dna with calcium chloride, adding this mixture in a. Give that jurkat is not an adherent cell line, and electroporation protocol is more. Jurkat cell line clone e61 was kindly provided by dr. Mouse t lymphocytes were obtained from total lymph node ln removal except mesenteric ln of c57bl6, balbc or b10a mice provided by incas animal facility. I use 24well plates for transfections in triplicates. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. One day prior to transfection, the cells were seeded in a 24well plate at a cell density of 12.
Cell cycle status and mitotic activity modulate the competence of target cells for liposomal transfection brunner et al. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines. Transfection guide overview of transfection methods promega. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. This reference provides a recommended procedure to transfect plasmid dna into human jurkat human t cell leukemia cells atcc cat. Wash the cells 2x pbs seed 10 6 cells in 300 ul warm optimem in 24 wellsplates. What is the best method to transfect tcells jurkat. Lipofectamine 3000 reagent transfection efficiency.
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