Transfection of thp 1 cells use this procedure to transfect plasmid dna into thp 1 cells in a 24well format for other formats, see scaling up or down transfections, below. All amounts and volumes are given on a per well basis. Please refer to table 1 for recommended reaction conditions for other dish or plate sizes. Viral transfection of thp1 cells has been described lee et al. Lipofectamine 2000 transfection protocol 10cm plates reagents. For additional information on scaling your transfection reaction, see page 4. The products included in this piece are our most robust transfection reagents that provide solutions to both common and hardtotransfect cell types, promoting effective. Differentiated thp1 cells were treated with 60 nm sirna. Lung, chang gung memorial hospital, chiayi, tw data from m. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Im trying to obtain a silencing in thp 1 cell using commercial sirna after which i have to stimulate my cell to induce cytokine production. Knockdown of myd88 and ap2 in thp1 monocytes after sirna transfection with. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates.
The addition of antibiotics to media during transfection may result in cell death, and has not been tested for thp 1 cells. Invitrogen lipofectamine 2000 transfection reagent. Therefore, most studies using transfected thp 1 cells avoid maturation. The addition of antibiotics to media during transfection may result in cell death, and has not been tested for thp1 cells. Therefore a transfection protocol is required that is capable of transferring sirna and plasmid dna into macrophages without causing serious. Im trying to obtain a silencing in thp1 cell using commercial sirna after which i have to stimulate my cell to induce cytokine production. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Human monocytic cell line aml reduction of aha1 mrna expression in raw macrophages upper and thp1 monocytes bottom by sirna transfection with viromer blue. Cells should be plated on collagencoated dishes, as for normal propagation. Bgov1, human embryonic stem cells, plasmid dna, lipofectamine 3000. Lipofectamine 2000 reagent thermo fisher scientific. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. For thp1 and u937 cells, no transfection was observed no green fluorescent cells. 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 maintaining high cell vitality and full macrophage capacity for differentiation and polarization.
Clear distinctions in the transfection efficiency between the ten cell types are seen. 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. Viromag rl is a magnetic beads containing formulation specifically designed for retrovirus and lentivirus, which allows to increase your transduction efficiency. In tube a place 2 g of dna in optimemi for a final volume of 200 l. However, thp1 cells are notoriously difficult to transfect. Although invitrogen recommends their own brand of culture medium for transfection, called optimem, we found no difference between this and conventional media. Plate cells so they will be 7090% confluent at the time of transfection. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity.
Before transfection, i have the need to differentiate thp1 they became adherent to induce receptor on cell surface and only after this passage i can transfect the cells. Dilute appropriate amount of lipofectamine 2000 by optimem i reduced serum medium to 50 l and vortex for a few seconds for siha and skov3 cells, dilute 1. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Invitrogen lipofectamine 3000 transfection reagent l3000008. Invitrogen lipofectamine transfection reagent protocols have been optimized for. Examiningransfection the ttes ofra thp1 cellsing by utiliz metafectene and lipid f. Transfection of thp1 cells with plasmids or small interfering rna sirna is achieved by electroporation using the lonza nucleofector technology basel, switzerland. For thp 1 and u937 cells, no transfection was observed no green fluorescent cells.
A method for high transfection efficiency in thp1 suspension. Lipofectamine 2000 dna jetmessenger outperforms its main competitor. Lipofectamine 2000 is then added to culture medium within a sterile tube along with the dna at a 3. Lipofectamine stem reagent maintains healthy, proliferating stem cells during transfection. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Transfections were performed on several cell lines following standard protocol. I use 24well plates for transfections in triplicates. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent optional 0. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. Okita in the laboratory of professor yamanaka at the center for ips cell research and application cira, kyoto university. Highly efficient transfection of human thp1 macrophages.
Maleszewska, nencki institute of experimental biology warsaw, pl. The following protocol describes how to transfect plasmid dna into thp1 cells using the genexplus reagent in a 6 well plate. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Notably, nate is gentle on cells during the transfection protocol data not shown. Transfection is a useful tool to study the function of gene products, but transfection of thp1 monocytes and predifferentiated thp1 macrophages with subsequent differentiation into mature thp1 macrophages using phorbol esters is usually accompanied by a progressive loss of cell viability. Add 6l of lipofectamine2000 from biostores and 100 l of. The use of lipofectamine reagent for dna transfection in thp 1 or myeloma cells, and the use of other transfection reagents such as fugene 6 promega for dna transfection in thp 1 cells also showed similar transfection efficiency data not shown. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Highly efficient transfection of human thp1 macrophages by. In my experience, thp 1 transfection differentiates to type 2 macrophages induced by ltx, but i recommend it too. Lipofectamine 3000 reagent can deliver dna into dif.
Nonviral transfection of thp 1 monocytes has also been described martinet et al. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Hi susie, if youre trying to transduce thp 1 cells using lentivirus, i advise you to use viromag rl, which is supplied by ozbiosciences. In this study we used two commercial transfection reagents turbofect and lipofectamine 2000, to determine the possibility for transfection of the serumfree cell line mccoyplovdiv. The following protocol describes how to transfect plasmid dna into thp 1 cells using the genexplus reagent in a 6 well plate.
The transfection rates of thp1 cells ear compared using lipid f theiddle m panel and metafectene the bottom panel. This reference provides a recommended procedure to transfect plasmid dna into thp1 human monocytic leukemia cells atcc cat. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100. Improved protocol for efficient nonviral transfection of premature thp 1 macrophages. For each transfection sample, prepare dnalipofectamine 2000 complexes as follows. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Thp1 transfection with viromer viromer transfection. In my experience, thp1 transfection differentiates to type 2 macrophages induced by ltx, but i recommend it too. Transfection was measured by luciferase activity using a conventional assay. Improved protocol for efficient nonviral transfection of. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2.
Transfection protocol lipofectamine 3000 transfection. Prewarm 50ml of optimum stored in cold room at 4c 10min. The day of transfection, count the cells to determine culture density. The mechanism of lipofectamine 2000 mediated transmembrane. This protocol presents an efficient and reliable method to transfect human thp 1 macrophages with sirna or plasmid dna by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.
Pool dishes of cells that have been prepared for transfection. I am trying to transfect thp1 cell line using with lipofectamine. Higher densities can lead to spontaneous differentiation. Transfection of thp 1 cells with plasmids or small interfering rna sirna is achieved by electroporation using the lonza nucleofector technology basel, switzerland. Viromer green works extremely efficient in the sirna transfection of fibroblasts, colon carcinoma or in thp1 transfection.
Improved protocol for efficient nonviral transfection of premature thp1 macrophages. To transfect cells with sirna, follow the protocol as. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Highest transfection efficiency in many cell types and formats. Before transfection, i have the need to differentiate thp 1 they became adherent to induce receptor on cell surface and only after this passage i can transfect the cells. The most effective transfection results were obtained with hela, huh7, modek and mg63 cell lines with both nanoparticles and lipofectamine. Nate validation data sheet transfection reagent invivogen.
Maintain p19 cells undifferentiated in mem with 10% serum 7. Knockdown of myd88 and ap2 in thp 1 monocytes after sirna transfection with viromer green. Transfection protocol transfection components component cat. Highest transfection efficiency in many cell types and formats e. Thp1 cells were treated with lipofectamine 2000 mock transfection, sirna, or nonspecific rna pool, at the concentrations indicated. Dilute dna in 250 l of optimem i reduced serum medium without serum or other medium without serum. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Follow these important guidelines when transfecting dna into thp1 cells using lipofectamine ltx reagent. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Does anyone have recommendation for thp1 transfection. Knockdown of myd88 and ap2 in thp1 monocytes after sirna transfection with viromer green. Viafect transfection reagent transfection assay e4981. I can help if says about you need make in transfection. Polyplustransfection 2019 brochure chemie brunschwig.
This reference provides a recommended procedure to transfect plasmid dna into thp 1 human monocytic leukemia cells atcc cat. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng final lipofectamine 2000 reagent per well 0. Lipofectamine 2000 from invitrogen biocompare product. The vector contained an ofp reporter gene and was transfected. I have successfully nucleofected but would like to try transfection. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. 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. Prepare plasmid dnalipid complexes recommend 2 doses of lipid.
Genexplus transfection of plasmid dna into thp1cells. Optimizing transfection lipofectamine 2000 cd reagent. Establishment and characterization of a human acute monocytic leukemia cell line thp1. Follow these important guidelines when transfecting dna into thp 1 cells using lipofectamine ltx reagent. Preparation of the cells for transfection the day of transfection.
This transfection reagent is especially developed for the binding of smaller oligonucleotides that are used in the rnai technology like sirna and the regulation of protein expresson via mirna. May 31, 2009 transfection is a useful tool to study the function of gene products, but transfection of thp 1 monocytes and predifferentiated thp 1 macrophages with subsequent differentiation into mature thp 1 macrophages using phorbol esters is usually accompanied by a progressive loss of cell viability. Transfection is a useful tool to study the function of gene products, but transfection of thp 1 monocytes and predifferentiated thp 1 macrophages with subsequent differentiation into mature thp 1 macrophages using phorbol esters is usually accompanied by a progressive loss of cell viability. Lipofectamine 3000 reagent thermo fisher scientific. Have a look to users feedback on the attached pdf, and please contact me if you want. Cellspecific transfection protocols thermo fisher scientific us. Transfecting plasmid dna into thp1 cells using lipofectamine. Thp1, human monocytic leukemia, sirna, lipofectamine rnaimax, 778075. Aha1 sirna and control sirna as filled and open symbols, respectively. Efficient nonviral transfection of thp1 cells sciencedirect.
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