In the human monoctye cell line, thp1, the use of nate clearly increases. Sep 02, 2014 thp 1 macrophages were differentiated with 10 ngml pma for 48 hr and transfected with fluorescently labeled alexa 488 unspecific control sirna. Lipofectamine 2000 is sensitive to changes in temperature. Human monocytic cell line aml reduction of aha1 mrna expression in raw macrophages upper and thp1 monocytes bottom by sirna transfection with viromer blue. If you wish to use antibiotics during transfection, test your conditions thoroughly. Thp1, human monocytic leukemia, sirna, lipofectamine rnaimax, 778075. Viromer blue lipofectamine 2000 ctrl sirna sirna1 sirna2 l1236 l428 uho1 relative m r n a expression fold change comparison of knockdown efficiency in a selfestablished melanoma cell line after transfection of control and 2 target sirna with viromer blue and lipofectamine 2000. The following protocol describes how to transfect plasmid dna into thp1 cells using the genexplus reagent in a 6 well plate. This technique allows for the efficient nonviral delivery of plasmids, dna, rna, or sirna into primary cells or cell lines even if the cells are not or are only slowly proliferating. 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. Transfection of thp 1 cells with plasmids or small interfering rna sirna is achieved by electroporation using the lonza nucleofector technology basel, switzerland.
A method for high transfection efficiency in thp1 suspension. B and c, western blot analyses of class i a pi3k p110 catalytic subunit isoforms. Transfections were performed on several cell lines following standard protocol. Plate cells so they will be 7090% confluent at the time of transfection. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Follow these important guidelines when transfecting dna into thp1 cells using lipofectamine ltx reagent. Using this protocol, we have successfully transfected aons into the following cell lines.
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. All amounts and volumes are given on a per well basis. Viromer green works extremely efficient in the sirna transfection of fibroblasts, colon carcinoma or in thp1 transfection. Bgov1, human embryonic stem cells, plasmid dna, lipofectamine 3000. Thp1 cells treated only with pma for 24 h were designated m0 macrophages 53. The renilla luciferase vector was cotransfected with angptl4 deletion constructs and ror for control of transfection efficiency. 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. Thp1 macrophages were differentiated with 10 ngml pma for 48 hr and transfected with fluorescently labeled alexa 488 unspecific control sirna. The ysk12mend was transfected to human immune cell lines at a sirna dose range of nm, resulting that maximum gene silencing efficiencies at. It may also refer to other methods and cell types, although other terms are often preferred.
Hi susie, if youre trying to transduce thp1 cells using lentivirus, i advise you to use viromag rl, which is supplied by ozbiosciences. Pcsk9 sirna suppresses the inflammatory response induced. 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. On the day of transfection, 500 ng cas9 protein and 120 ng hprt1 grna were transfected with either lipofectamine 2000 lf2k, lipofectamine 3000 lf3k, lipofectamine messengermax, lipofectamine rnaimax, turbofect or xfect transfection reagent. Efficient mrna delivery in difficulttotransfect cells. Preparation of the cells for transfection the day of transfection. The lipofectamine 2000 and sirna solution were separately prepared in 1. Stable gene silencing in human monocytic cell lines using. Thp1 cell transduction transfection and transduction. A typical forward transfection protocol using transit2020 transfection reagent can be found here. Transfecting plasmid dna into thp1 cells using lipofectamine. Bovine nramp1, the homologue of a murine gene bcg, has been identified as a major candidate for controlling the in vivo resistant phenotype. For suspension cells andor high throughput applications, a reverse transfection protocol where freshly passaged cells are added to preplated transfection complexes is ideal as it reduces handson time for the end user. Human monocytic cell line aml primary macrophages were freshly isolated from buffy coat pbmc and.
Pdf version aon transfection with lipofectamine 2000 3000. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Differentiated thp1 cells were treated with 60 nm sirna. Viromag rl is a magnetic beads containing formulation specifically designed for retrovirus and lentivirus, which allows to. Smallsized, stable lipid nanoparticle for the efficient. Comparison of transfection efficiency of thp1 monocytes by. Im trying to obtain a silencing in thp1 cell using commercial sirna after which i have to stimulate my cell to induce cytokine production. The alliance of mtx transfection reagent and combimag leads to increased transfection efficiency, minimized toxicity and enhanced gene expression. Transfection of thp1 cells with plasmids or small interfering rna sirna is achieved by electroporation using the lonza nucleofector technology basel, switzerland.
The mean fluorescence intensity mfi for mockinfected cells was 8. Thp1 cells were treated with lipofectamine 2000 mock transfection, sirna, or nonspecific rna pool, at the concentrations indicated. 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. Viafect transfection reagent transfection assay e4981. Briefly, prior to transfection differentiated thp1 cells 2.
Cells were either untreated or treated with 10 nm tpa 12 h after transfection and then. Nate validation data sheet transfection reagent invivogen. Pcsk9 sirna suppresses the inflammatory response induced by. Please refer to table 1 for recommended reaction conditions for other dish or plate sizes. However, thp1 cells are notoriously difficult to transfect. 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. We support multiple transfection methods with a different reagent for nearly any application. Viromag rl is a magnetic beads containing formulation specifically designed for retrovirus and lentivirus, which allows to increase your transduction efficiency. The transfection of sirna into thp1derived macrophages in suspension was conducted using lipofectamine 2000 according to the manufacturers instructions invitrogen, carlsbad, ca, usa. Apol1 renal risk variants induce aberrant thp1 monocyte. Gene expression genome editing genome editing protein delivery u n. Gene knockdown was carried out using trilencer27 sirna pool against mincle or a scrambled sirna control origene technologies, md. Lipofectamine 2000, fugene 6, sofast transfection reagents. Viral transfection of thp1 cells has been described lee et al.
We developed an in vitro model for expression of resistance and susceptibilityassociated alleles of bovine nramp1 as stable. Efficient nonviral transfection of thp1 cells sciencedirect. Cellspecific transfection protocols thermo fisher scientific us. Transfection transit transfection reagents mirus bio. Aha1 sirna and control sirna as filled and open symbols, respectively.
Knockdown of myd88 and ap2 in thp1 monocytes after sirna transfection with. The addition of antibiotics to media during transfection may result in cell death, and has not been tested for thp1 cells. Dna, sirna, mirna, mrna, viral rna, crisprcas9 components, and oligonucleotides to eukaryotic cells. Regulation of erkmediated signal transduction by p38 map. Transfection of thp1 cells use this procedure to transfect plasmid dna into thp1 cells in a 24well format for other formats, see scaling up or down transfections, below. Detection of nfb activation of the p50 and p65 subunits of nfb in macrophages was.
On the day of transfection, 500 ng cas9 protein and 120 ng hprt1 grna were transfected with either lipofectamine 2000 lf2k, lipofectamine 3000 lf3k, lipofectamine messengermax, lipofectamine rnaimax, turbofect or xfect transfection. Nextly, the mixed solution was added to thp1 cells to a final sirna concentration of 50 nm and thp1 cells were incubated. Transfection of dna by lipofectamine in cells has been used as a powerful tool for identification of the biological function of genes. Therefore a transfection protocol is required that is capable of transferring sirna and plasmid dna into macrophages without causing serious. For thp1 and u937 cells, no transfection was observed no green fluorescent cells. Does anybody have any experience with transfecting thp. I have attached the transfection protocol in primary. Notably, nate is gentle on cells during the transfection protocol data not shown. Highly efficient transfection of human thp1 macrophages. Lipofectamine 2000 dna jetmessenger outperforms its main competitor. This kit replaces and outperforms the original magnetofectamine which was the combination of lipofectamine 2000 from life technologies corporation and combimag transfection reagent. Lipofectamine 2000 reagent thermo fisher scientific. Hi susie, if youre trying to transduce thp 1 cells using lentivirus, i advise you to use viromag rl, which is supplied by ozbiosciences. Automated highthroughput sirna transfection in raw 264.
The day of transfection, count the cells to determine culture density. The most effective transfection results were obtained with hela, huh7, modek and mg63 cell lines with both nanoparticles and lipofectamine. 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. Improved delivery of cas9 proteingrna complexes using. Knockdown of myd88 and ap2 in thp1 monocytes after sirna transfection with viromer green. In contrast to most chemical transfection approaches, our modified nucleofection procedure using premature macrophages yields high transfection efficiencies in combination with unimpaired cell. Have a look to users feedback on the attached pdf, and please contact me if you want. Transfection was performed using lipofectamine 2000 reagent following the manufacturers instructions invitrogen, gaithersburg, md. This approach allows for the transfection of cell lines such as human thp1 monocytes and macrophages and has been successfully applied in the past 610.
Boese,3 peter sommer,3 auguste genovesio,2 and priscille brodin1 rnai using sirna is a very powerful tool for functional genomics to identify new drug targets and biological pathways. A type i ifn levels in supernatants from pmadifferentiated thp1 cells transfected with lipofectamine 2000 with pcrderived dsdna of indicated lengths at 1. I am trying to transfect thp1 cell line using with lipofectamine. Download the easytouse quick guide or the full transfection protocol here and prepare for your next thp1 transfection experiment. Thp1 jurkat primary mes fibroblasts jetmessenger mrna lipofectamine 2000 dna inde l % 30 60 20 50 10 40 0 a549 hek293 jetcrispr lipofectamine crisprmax request your sample of jetprime, jetcrispr, jetmessenger or interferin on. However, thp 1 cells are notoriously difficult to transfect. Transfection efficiency % 80 100 60 40 20 0 thp1 jurkat primary mes fibroblasts jetmessengermrna lipofectamine 2000 dna indel % 30 60 20 50 10 40 0 a549 hek293 jetcrispr lipofectamine crisprmax request your sample of jetprime, jetcrispr, jetmessenger or interferin on. The transfection rates of thp1 cells ear compared using lipid f theiddle m panel and metafectene the bottom panel. If you wish to use antibiotics during transfection, test.
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. To select the most effective sirna concentration, reverse transcriptionpolymerase chain reaction rtpcr and western blot analysis were performed 24 h. For luciferase assay, transfections were performed with lipofectamine 2000 transfection reagent invitrogen, 11668019 in hek293t cells. The transfection of mrna was performed using three commercially available transfection reagents including lipofectamine messengermax thermo scientific, viromer red.
Dephosphorylation of the rna sensors rigi and mda5 by the. 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. Nov 28, 2016 the ysk12mend was transfected to human immune cell lines at a sirna dose range of nm, resulting that maximum gene silencing efficiencies at the mrna level in jurkat, thp1, kg1 and nk92. Invitrogen lipofectamine transfection reagent protocols have been optimized for. In animal cells, transfection is the preferred term as. Stable transfection of the bovinenramp1 gene into murine. The products included in this piece are our most robust transfection reagents that provide solutions to both common and hardtotransfect cell types, promoting effective. A case study for optimization procedure jeanphilippe carralot, 1 taekyu kim, boris lenseigne,2 annette s.
To select the most effective sirna concentration, reverse transcriptionpolymerase chain reaction rtpcr and western. Our reagents are ideal for delivering multiple types of nucleic acids including. Efficient mrna delivery in difficulttotransfect cells with. For thp 1 and u937 cells, no transfection was observed no green fluorescent cells. Highly efficient transfection of human thp1 macrophages by. Thp 1 0,01 0, 1 1 10 100 0 50 150 100 human primary macrophages pbmcs promyoblast macrophage cell line kg1a % of target m r n a nm sirna ec 50 0. The following protocol describes how to transfect plasmid dna into thp 1 cells using the genexplus reagent in a 6 well plate. Examiningransfection the ttes ofra thp1 cellsing by utiliz metafectene and lipid f. Clear distinctions in the transfection efficiency between the ten cell types are seen. Improved protocol for efficient nonviral transfection of.
Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. Regulation of erkmediated signal transduction by p38 map kinase in human monocytic thp1 cells. Genexplus transfection of plasmid dna into thp1cells. Viral transfection of thp 1 cells has been described lee et al. Magnetofectamine o2 transfection kit oz biosciences. To transfect cells with sirna, follow the protocol as. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
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