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. All amounts and volumes are given on a per well basis. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Component 96well 24well 6well final dna per well 50100 ng 250500 ng 1. Thp1 cells, purchased from the korean cell line bank, were cultured in rpmi. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000.
Dna complexes were added to cells that had already been allowed to attach to the plate b. Lipofectamine 3000 is a new transfection reagent which is claimed to be more efficient than other transfection reagents like turbofect. This was further increased upon treatment with ed64 and. Does anybody have any experience with transfecting thp.
The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and. Does anyone have recommendation for thp1 transfection. Make sure that cells are greater than 90% confluent and vary dna. Dna complexes were added to wells so that transfection could occur as cells were attaching to the plate. Introducing the versatile reagent with the power to transfect.
Mar 01, 2012 transfection of the sirna oligomers will be performed using lipofectamine 2000. Thp1 cells will be treated with lipofectamine 2000 mock transfection, sirna, or nonspecific rna pool. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Dharmafect transfection reagentssirna transfection. We have been successful in the past with transfecting thp1 cells in floating and in. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Add 1g of bac dna to each tube and mix by flicking tube 4. Transfection efficiency was measured using fluorescence microscope. Values were normalized to transit2020 and presented as a percentage of luciferase expression. Genexplus transfection of plasmid dna into thp1cells. Using lipofectaminez 2000 for transfection provides the following advantages.
However, thp1 cells are notoriously difficult to transfect. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Successful transfection of thp1 monocytes with subsequent phorbol 12myristate acetate pmainduced differentiation into macrophages is not a trivial matter, because according to previous transfection protocols, cell viability is lost almost completely within 24 h of pma. Rinse cells 1x w pbs, add fresh media and return to 37c make sure media is warm else cells will detach 7. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Cells were either untreated or treated with 10 nm tpa 12 h after transfection and then incubated for another 12 h. Transfecting plasmid dna into thp1 cells using lipofectamine. The following is a general protocol for use of dharmafect transfection reagents to deliver sirna into cultured mammalian cells. Lipofectamine 2000 is sensitive to changes in temperature. The mechanism of lipofectamine 2000 mediated transmembrane. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
Comparison of transfection efficiency of polymerbased and. Dharmafect transfection reagentssirna transfection protocol. Do not add antibiotics to media during transfection as this causes cell death. The addition of antibiotics to media during transfection may result in cell death, and has not been tested for thp1 cells. Successful transfection of thp1 monocytes with subsequent phorbol 12myristate acetate pmainduced differentiation into macrophages is not a trivial matter, because according to previous transfection protocols, cell viability is lost almost completely. For thp 1 and u937 cells, no transfection was observed no green. Transit2020 transfection reagent research reagent products. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in a 96well format, and. To date, how to reach the maximum transfection and viral transduction efficiency is still challenging. Feb 18, 2016 identification of lipofectamine crisprmax. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Cell type, source, application, recommended transfection reagent, recommended. Examiningransfection the ttes ofra thp1 cellsing by utiliz metafectene and lipid f. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium.
Efficient nonviral transfection of thp1 cells sciencedirect. The day of transfection, count the cells to determine culture density. 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. Lipofectamine rnaimax has a broad peak of activity. An improved method for increasing the efficiency of gene. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. By mixing dna with lipofectamine 2000, positively charged liposomes. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment.
Maintain p19 cells undifferentiated in mem with 10% serum. 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. Please refer to table 1 for recommended reaction conditions for other dish or plate sizes. The intracellular trafficking mechanism of lipofectamine. Highly efficient transfection of human thp1 macrophages by. Collect viruscontaining medium 48 hours after transfection and replace with fresh medium.
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. Scaling up or down lipofectamine 3000 reagent transfections. The human monocytic leukemia cell line thp1 is a widely used model for investigating monocyte and macrophage biology. Lipofectamine 3000 is the best choice for transfection of chok1 and hek293 with pcdh while turbofect is preferably used in transfecting these cell lines with pegfpn1 tab. In my experience, thp1 transfection differentiates to type 2 macrophages induced by ltx, but i recommend it too. Follow these important guidelines when transfecting dna into thp1 cells using lipofectamine ltx reagent. 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. Does anybody have any experience with transfecting thp floating. For hek293t cells we find that 1520 minutes is optimal. A sample protocol is listed here for transfection experiments performed in 6well plates. Add the dna cocktail transfection buffer mix dropwise to the cells some protocols recommend waiting 1530mins for precipitate to form but i get better transfection when i dont wait 5.
Jun 01, 2004 a transfection of griptite293 msr cells with a. Here we compared the transfection and viral transduction efficiency using commercially available transfection reagents including fugene 6, lipofectamine 2000 and lipofectamine 3000 in different cell lines and primary cultured cells. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. Advanced transfection with lipofectamine 2000 reagent. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. Lipofectamine 3000 lipofectamine 2000 fugene hd lipofectamine 2000 lipofectamine 3000 competitor 1 competitor 2 western blot, hepg2 gststat. Data were collected from three independent experiments. Thp1 cells will be transfected with sirna using the nucleofector solution v reagent amaxa according to the protocol provided by the. Therefore a transfection protocol is required that is capable of transferring sirna and plasmid dna into macrophages without causing serious. Lipofectamine 3000 reagent thermo fisher scientific jp. Thp1 cells were transfected with ifi16bflag plasmid. For transfecting thp 1 cells should you use lipofectamine 3000 or ltx. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1.
Bgov1, human embryonic stem cells, plasmid dna, lipofectamine 3000. Transfected dna of bacterial, viral, and mammalian origin causes formation of the inflammasome in murine macrophages and in the human monocyte cell line thp1 muruve et al. The following protocol describes how to transfect plasmid dna into thp1 cells using the genexplus reagent in a 6 well plate. Transfection efficiency and protein expression using geneart crispr nuclease vector. Viral transfection of thp1 cells has been described lee et al. Automated highthroughput sirna transfection in raw 264. 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. Comparison of dnatransfection efficiency in shsy5y cells. The use of thp1 cells as a model for mimicking the. Lipofectamine 2000 cd reagent thermo fisher scientific. With lipofectamine, i have no reference, but with our polymerbased reagent viromer red, our customers achieved never more than 25% of transfected cells with thp1 monocytes.
Comparative transfection efficiency of jetprime versus its main competitor. Improved protocol for efficient nonviral transfection of. 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. I can help if says about you need make in transfection. I am trying to transfect thp1 cell line using with lipofectamine. Transfection selection tool thermo fisher scientific jp.
Plate cells so they will be 7090% confluent at the time of transfection. I have successfully nucleofected but would like to try transfection. Transfection protocol lipofectamine 3000 transfection. Luciferase activity was measured 24 hours post transfection.
Successful transfection of thp1 monocytes with subsequent phorbol 12myristate acetate pmainduced differentiation into macrophages is not a trivial matter, because according. L mrna transfection mrna can be transfected in a 24well plate with 1 or 2. If you wish to use antibiotics during transfection, test. Viafect transfection reagent transfection assay e4981. In this study, the optimal dose of lipofectamine 3000 and turbofect to transfect adherent cell lines such as chok1 and hek293 cells in comparison with nonadherent h9tcells with pegfpn1 and pcdh was identified. Thp1 differentiation and transfection easy or difficult. The reagent provides high transfection efficiency in many cells types and formats. Transfection amounts component 96well 24well 6well final mrna per well 100 ng 500 ng 2500 ng final lipofectamine. As shown in table 1, only lipofectamine ltx, lipofectamine. Have a look to users feedback on the attached pdf, and please contact me if you want. Optimizing transfection lipofectamine 2000 cd reagent. It may also refer to other methods and cell types, although other terms are often preferred. The transfection rates of thp1 cells ear compared using lipid f theiddle m panel and metafectene the bottom panel. Lipofectamine messengermax reagent protocol outline a.
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. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Have a look to users feedback on the attached pdf, and please contact. In animal cells, transfection is the preferred term as. Gibco hams f12 nutrient mix 11765054 5% horse serum 1 mm gibco sodium pyruvate 160070 25 mm gibco hepes 15630106 proper culture techniques and procedures are an essential. The optimal dharmafect reagent amount varies for different cell lines and is affected by the cell density. The inflammasome is a protein complex formed around one of multiple sensor proteins, member of the nodlike receptor nlr or pyhin. After differentiation of thp1 gfplc3 cells into macrophages by treatment with phorbol myristate acetate pma, we transfected polyda.
Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. Keep all viral media at 4c until all collections are done. Seed 500,000 pure k5 62 cells in 12 well plate, in 1ml complete rpmi media. The use of thp1 cells as a model for mimicking the function. Genscript recommends using lipofectamine 2000 for all transfections. Transfection protocol lipofectamine 3000 transfection reagent. I have attached the transfection protocol in primary. I tried this with lipofectamine 2000 in the past with almost no transfection as measured by flow. A case study for optimization procedure jeanphilippe carralot, 1 taekyu kim, boris lenseigne,2 annette s.
Gene knockdown was carried out using trilencer27 sirna pool against mincle or a scrambled sirna control origene technologies, md. The animal originfree formulation ensures that mammalian cell culture and bioproduction processes are free of animalderived materials. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Improved delivery of cas9 proteingrna complexes using. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. May 31, 2009 however, thp1 cells are notoriously difficult to transfect. Highest transfection efficiency in many cell types and formats. The highest transfection efficiency in many cell types and formats e. Different cell lines were transfected with a gfp reporter vector using the indicated transfection reagents. Dna transfection see page 2 to view a typical dna transfection procedure. Reagent to dna ratios were optimized for each cell type. Smallsized, stable lipid nanoparticle for the efficient.
To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as dna and lipofectamine 2000 concentrations. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. For luciferase assay, transfections were performed with lipofectamine 2000. Thp 1 cells, purchased from the korean cell line bank, were cultured in rpmi. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. Preparation of the cells for transfection the day of transfection. The examples given within the protocol are for 96well plates, and table 1 provides the.
Achieve high expression in difficult to transfect cells, including huvec, thp1, and mef. If you wish to use antibiotics during transfection, test your conditions thoroughly. Transitx2 dynamic delivery system mirus bio and lipofectamine 2000 transfection reagent thermo fisher scientific were used to. Lipofectamine 2000 invitrogen according to the manufacturers instructions and as. Lipofectamine 2000 reagent thermo fisher scientific.
Enhanced transfection efficiency using lipofectamine 3000 reagent. Transfection of thp1 cells with plasmids or small interfering rna sirna is achieved by electroporation using the lonza nucleofector technology basel, switzerland. Briefly, prior to transfection differentiated thp1 cells 2. I am trying to transfect thp 1 cell line using with lipofectamine. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. 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. Collect virus every 12 hours for up to 3 times total. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Cereblon suppresses the lipopolysaccharideinduced inflammatory.
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