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