Lipofectamine 3000 cho 32 μg) and LipofectamineTM 2000 Reagent (0. 6% with Turbofect and Lipofectamine 3000, respectively. Each Explore Lipofectamine 3000 transfection reagent, offering high-efficiency transfection and improved cell viability in the broadest range of cell types. Invitrogen Lipofectamine 3000 Transfection Reagent is our highest efficiency reagent for difficult-to-transfect cells. Each reaction mix volume is for one well and accounts for pipetting variations. 4. 1, Fig. I add 7ul of Lipofectamine 3000 to a separate tube with 125ul of opti-mem and vortex for three seconds. Lipofectamine® 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application. Hard-to-transfect cell lines were defined as having less than 40% transfection efficiency. H9T-cells show inadequate transfection rates, achieving only 0. Mar 5, 2021 · Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. The Cas9 RNPs in Opti-MEM medium were added to the transfection reagents diluted in Opti-MEM medium. I usually add p/s to DMEM (along with l-glu) and use it to transfect HEK293 cells using lipofectamine 3000 but I haven't been able to see any protein expression when analyzing the medium. Find additional tips, troubleshooting help, and resources within our Transfection Support Center. For transfection with Lipofectamine 3000 reagent (Figure 1), in separate tubes, 1. While lipofectamine may favor different conditions than other techniques, the same steps can be taken to optimize most known chemical transfections. , 2014). It consistently produces high transfection efficiency and high protein overexpression. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. * After determining the optimum reagent amount, use the multiplication factor to determine the reagent amount needed for your new plate format. Lipofectamine® LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. Start any new transfection by testing the recommended two concentrations of LipofectamineTM 3000 Reagent to determine an optimum amount. [1] It is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection. Transfection protocol Transfection components Component Cat. Lipofectamine ® 2000 and FuGENE ® HD have comparable transfection efficiencies but lower than that of Lipofectamine ® 3000. FuGENE® 4K DNA Transfection Outperforms Lipofectamine® 3000 in most cell types Most effective broad spectrum DNA transfection reagent Maximum transfection efficiency in adherent cells Robust performance in suspension adapted 293 & CHO cells Improved viral titers and protein production yields. The 3k probably had a slighly higher transfection efficiency. 75-7. Lipofectamine LTX Reagent is a plasmid transfection reagent that offers a balance of potency and gentleness for your cells, resulting in high transfection efficiencies and viabilities. Transfection efficiency comparison of various transfection reagents (PEI-Max, Transfectin, and Lipofectamine 3000). [1] Lipofectamine 3000 reagent is a specially formulated gentle transfection reagent that is optimized for plasmid delivery into CHO cells with high efficiency and low cell toxicity. Invitrogen Lipofectamine 3000 Transfection Reagent from Invitrogen™. 试剂和有效载荷复合物的形成 Lipofectamine 3000 试剂盒 Lipofectamine 3000试剂 P3000 试剂 DNA 您需要递送的DNA 附着和入胞 通过内吞作用 进入细胞 刺激内涵体 逃逸 促进DNA入核 结果：高效表达蛋白 蛋白质 内涵体 DNA mRNA 细胞核 Invitrogen™Neon™Transfection System 图4. The ExpiFectamine CHO Transfection Kit is a core component of the ExpiCHO Expression System, designed for transient transfection of high-density cultures of Chinese hamster ovary (CHO) cells. More importantly, Lipofectamine®3000 has the potential to help propel many of these novel and exciting technologies forward. Sep 28, 2016 · 2. HyCell™ CHO media are chemically defined and animal-derived component-free. FuGENE 4K Outperforms Lipofectamine®3000 in most cell types Most effective broad spectrum DNA transfection reagent Maximum transfection efficiency in adherent cells Robust performance in suspension adapted 293 & CHO cells Improved viral titers and protein production yields Use the indicated volume of DNA and P3000TM Reagent with each of the two volumes of LipofectamineTM 3000 (when performing optimization). Prepare master mix of DNA by diluting DNA in Opti-MEMTM Medium, then add P3000TM Reagent – Mix well. Incubate for 10–15 minutes at room temperature. If transfection efficiency is of concern, Lipofectamine 3000 is better than Lipofectamine May 5, 2019 · The amount of LipofectamineTM 3000 Reagent for successful transfection varies. More importantly, Lipofectamine® 3000 has the potential to help propel many of these novel and exciting technologies forward. 2 μl to 1. Optimum transfection efficiency and subsequent cell viability depend on a number of experimental variables such as cell density, liposome and DNA concentrations, liposome–DNA complexing Explore Lipofectamine 3000 transfection reagent, offering high-efficiency transfection and improved cell viability in the broadest range of cell types. 1 mL. Jan 1, 2018 · Objectives In this study, the optimal dose of Lipofectamine 3000 and Turbofect to transfect adherent cell lines such as CHO-K1 and HEK293 cells in comparison with non-adherent H9T-cells with pEGFP-N1 and pCDH was identified. InvitrogenTM LipofectamineTM 3000 Transfection Reagent was developed to unleash the power of stem cells by providing a highly efficient, cost Scaling up or down Lipofectamine 3000 reagent transfections Use the following table to scale the volumes for your transfection experiment. Results: Lipofectamine 3000 was more efficient in transfecting pCDH, while Turbofect was more efficient in separate transfection of CHO-K1 and HEK293 with pEGFP-N1. It is primarily used in molecular biology and gene therapy for research applications, allowing researchers to study gene expression, gene silencing, and other genetic manipulations. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection Explore Lipofectamine 3000 transfection reagent, offering high-efficiency transfection and improved cell viability in the broadest range of cell types. A variety of cell lines were seeded and subsequently transfected with GFP-expressing plasmid using FuGENE® 4K or Lipofectamine™ 3000 transfection reagent. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of CHO-K1 cells with a reporter plasmid. Hits were optimized and retested in expanded cell panels. 1 mL CHO-S cells were transfected with pCMV•SPORT-βgal DNA (0. What I generally do is : OBJECTIVES: In this study, the optimal dose of Lipofectamine 3000 and Turbofect to transfect adherent cell lines such as CHO-K1 and HEK293 cells in comparison with non-adherent H9T-cells with pEGFP-N1 and pCDH was identifi ed. GFP expression was analyzed 48 hours posttransfection. Lipofectamine 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible resul. Has anyone here attempted reverse transfection using Lipofectamine 3000 reagent? I am attempting to deliver a plasmid ~9000 bp in size into hard-to-transfect lung fibroblasts. We have used both Lipofectamine 2000 and Lipofectamine 3000 for this purpose in HEK293T and there wasn't that much difference. BACKGROUND: There is a major problem with transfections in our project. Lipofectamine™ 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. FuGENE® 4K reagent outperforms Lipofectamine™ 3000 across many cell lines. Twenty-four hours prior to transfection, 30 mL of FreeStyle™ CHO expression medium containing 6 × 10 5 cells/mL was seeded into 125 mL shaking flasks. Attached at the end of this protocol is the product information sheet and protocol guidelines from Gibco. The mixture was incubated at 25 °C for 10–15 min to form the Cas9 RNPs and transfection reagent complexes, followed by addition to the cells. Such formulation increases transfection efficiency of nucleotide molecules in both dividing and non-dividing cells under in vitro conditions. Figure 2. GenScript recommends using Lipofectamine 2000 for all transfections. Aug 20, 2015 · Lipofectamine 3000 and RNAiMAX outperformed Lipofectamine 2000 in HEK 293 cells (Figure S1), which is in agreement with the recent finding that RNAiMAX performed better than Lipofectamine 2000 for delivery of Cas9 RNPs with low cell toxicity (Zuris et al. Sep 1, 2020 · Fig. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. More importantly, Lipofectamine 3000 reagent has the potential to help propel many of these novel and exciting technologies forward. Cells were evaluated for transfection efficiencies after 48 hours post-transfection. Oct 9, 2023 · Lipofectamine 3000 transfection protocol Day 0: Cells are grown in 12-well, 24-well plates with coverslips gelatinized to the bottom of LipofectAMINE is purchased from Gibco-BRL. Lipofectamine 3000 shouldn't be cytotoxic especially to CHO cells. These CHO cell culture media are designed for high yield of CHO cells and for recombinant protein production. 5μL LipofectamineTM 3000 Reagent in 250μL Opti-MEMTM Medium (2 tubes) . No. Generation and transfection of induced pluripotent stem cells (iPSCs) Induced pluripotent stem cells (iPSCs) hold immense promise for the future of regenerative medicine and personalized therapeutic treatments for a myriad of diseases and conditions. Add Diluted DNA to each tube of Diluted LipofectamineTM 3000 Reagent (1:1 ratio). Expi293F, CHO, A549, NIH-3T3, COS-7, HepG2 cells were transfected with a GFP-expressing plasmid using LipoFectMax™, LipoFectMax™ 3000, Lipofectamine® 2000 and Lipofectamine® 3000 transfection reagent, respectively. 7% and 1. Scale volumes proportionally for additional wells. I’ve attempted to transfect NIH/3T3 cells using Lipofectamine 3000 and I‘ve achieved around 10-20% of transfection. Figure 6. LipofectamineTM 3000 Reagent Protocol Transfect cells according to the following table. CONCLUSION: Lipofectamine 3000 is the best choice for transfection of CHO-K1 and HEK293 with pCDH while Turbofect is preferably used in transfecting these cell lines with pEGFP-N1 (Tab. 脂質を介した DNA のデリバリー Lipofectamine 3000 Transfection Reagent with P3000 Reagent InvitrogenTM LipofectamineTM 3000 Transfection Reagent with P3000TM Reagent は、困難な細胞をトランスフェクションする力を提供する、DNA デリバリーのための当社が 最もおすすめする製品です。 I use lipofectamine 2000 reagent for transfection. Can I use Lipofectamine RNAiMAX to co-transfect siRNA with plasmid DNA? While Lipofectamine 3000 or Lipofectamine 2000 may be used for co-transfection of siRNA with plasmid DNA, Lipofectamine RNAiMAX cannot be used. CHO-S cells were transfected with pCMV•SPORT-βgal DNA (0. In Lipofectamine 3000, cationic lipid molecules (DOSPA and DOPE) are formulated with a neutral/helper co-lipid (P3000 reagent). Feb 18, 2016 · Identification of Lipofectamine CRISPRMAX To identify the best transfection reagents for delivery of Cas9 RNPs, initially we compared several commercially available protein transfection reagents, including Lipofectamine 2000, Lipofectamine 3000, Lipofectamine RNAiMAX, Lipofectamine MeassengerMax, TurboFect, and Xfect Protein transfection reagent. The transfection efficacy of Lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. Explore Lipofectamine 3000 transfection reagent, offering high-efficiency transfection and improved cell viability in the broadest range of cell types. Lipofectamine® RNAiMAX works best at confluencies between 60 and 80%. I wait about 5 minutes, then add the two solutions together. 5 mL Lipofectamine™ 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types More … In another study that involved human bone marrow mesenchymal stem cell (hBM-MSC), Lipofectamine LTX was shown to produce the best transfection efficiency (at least three times higher) than other reagents such as TransIT-2020, Lipofectamine 3000 and polyethylenimine (PEI) but presented low cell viability (<50%) (Cheung et al. Features Supplier: Invitrogen™ L3000015 Lipofectamine™ 3000 Transfection Reagent 1. The amount of Lipofectamine® LTX Reagent required for successful transfection varies depending on the cell type and passage number. Abstract Lipofectamine reagents are widely accepted as “gold-standard” for the safe delivery of exogenous DNA or RNA into cells. Learn about CHO-K1, a subclone derived from the parental CHO cell line. Optimal transfection conditions include 1 Jul 9, 2015 · I find the protocol for lipofectamine 3000 very confusing with the 2 reagents that have a very similar name! on top of that the insert shows an example for 2 wells instead of one. View the resources available to support their growth in many research applications. Lipofectamine® 3000, Lipofectamine® 2000, and Lipofectamine® LTX/PLUS provide excellent transfection performance at confluencies between 70 and 90%, while some toxicity may be observed at confluencies lower than this. Aug 20, 2025 · Lipofectamine is available in multiple different formats, including Lipofectamine 2000, Lipofectamine 3000, Lipofectamine RNAiMAX and Lipofectamine LTX. Lipofectamine® 3000 demonstrates signifi cant improvement in a broader spectrum of cell lines when compared to current commercially available lipid-mediated transfection reagents. Lipofectamine™ 3000 Transfection Reagent. I usually leave the cells in complete media use the optimem for the lipofectamine dilution and add dropwise in a sort of lollipop swirl. This reagent provides an advanced solution for gene expression in common cell lines, particularly Chinese Hamster Ovary (CHO) cells, and challenging cell types such as primary neural progenitor cells, primary Lipofectamine 3000 reagent (Life Technologies, Carlsbad, CA, USA) was used to co-transfect the FreeStyle CHO-S cells in triplicate. I am using 6 well plates for the experiment. 1 Comparison of Lipofectamine™ 2000, Lipofectamine™ 3000 and Transfectamine™ 5000 using various platforms. A sample protocol is listed here for transfection experiments performed in 6-well plates. HeLa cells and CHO-K1 cells were transfected with a GFP expressing plasmid using Lipofectamine™ 2000, Lipofectamine™ 3000 and Transfectamine™ 5000 following their corresponding protocols. LipofectamineTM 3000 Reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells and especially designed for hard to ∤ transfect cells Make DNA-LipofectamineTM 3000 complexes in serum-free medium such as Opti-MEMTM Reduced Serum Medium and add directly to cells in culture mediu On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24-well plate using Lipofectamine 3000 Transfection Reagent: Lipofectamine®3000 demonstrates signifi cant improvement in a broader spectrum of cell lines when compared to current commercially available lipid-mediated transfection reagents. 293FT cells were transfected with various DNA transfection reagent complexes Lipofectamine™ Transfection Reagent is one of our first-generation, cationic-lipid transfection reagents formulated for the transfection of DNA into eukaryotic cells. On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24-well plate using Lipofectamine 3000 Transfection Reagent: Feb 14, 2025 · What is Lipofectamine and How Does It Work? Lipofectamine is a lipid-based transfection reagent designed to facilitate the delivery of genetic material, such as DNA, RNA, or siRNA, into cells. Start any new transfection by testing the recommended four concentrations of Lipofectamine® LTX Reagent to determine an optimum amount. Lipofectamine has May 11, 2016 · We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules and the subsequent entrapment and degradation of Lipofectamine 3000 reagent demonstrates significant improvement in a broader spectrum of cell lines when compared to current commercially available lipid-mediated transfection reagents. Lipofectamine 3000 demonstrates higher transfection efficiency for pCDH, while Turbofect is superior for pEGFP-N1. IDK what everyone else is on about it's one of the least cytotoxic transfection reagents in vitroconfluency (and proper math) matters though. On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24-well plate using Lipofectamine 3000 Transfection Reagent: Explore Lipofectamine 3000 transfection reagent, offering high-efficiency transfection and improved cell viability in the broadest range of cell types. This is often the best place to start, especially in a new cell line. Our Lipofectamine Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Passage number may affect transfection experiments. If you find this doesn’t work for your specific cell type, then you can you look to our cell specific protocols for further optimization. Use the indicated volume of DNA and P3000TM Reagent with each of the two volumes of LipofectamineTM 3000 (when performing optimization). Invitrogen™Lipofectamine 3000 Transfection Reagent L3000008 Gibco™Opti-MEM I Reduced Serum Medium 31985062 Thermo Scientific™Nunc 24-Well Cell-Culture Treated Multidishes 142475 Step Tube Complexation components Amount per well (24-well) Explore Lipofectamine 3000 transfection reagent, offering high-efficiency transfection and improved cell viability in the broadest range of cell types. Jan 1, 2019 · Dilute 3. 2 μl, columns 1-6 respectively) in 96-well plates. Jul 19, 2016 · In general, Lipofectamine ® 3000 outperforms Lipofectamine ® 2000 and FuGENE ® HD in both transfection efficiency (A,B) and protein expression (C). 5 µg plasmid/well and the recommended protocols for each reagent. Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. Mar 31, 2019 · Our experiments were conducted using mainly Lipofectamine 3000 and a single experiment of Calcium Phosphate based Transfection, so protocols and experimental data were made for these protocols. OBJECTIVES: In this study, the optimal dose of Lipofectamine 3000 and Turbofect to transfect adherent cell lines such as CHO-K1 and HEK293 cells in comparison with non-adherent H9T-cells with pEGFP-N1 and pCDH was identifi ed. 2 According to our results, Lipofectamine 3000, Fugene and RNAiMAX showed high transfection efficacy, however, RNAiMAX may be a better option for majority of cells when lower toxicity is desired. view related products. Therefore, our results suggest that a much higher transfection and viral transduction efficiency can be attained by using Lipofectamine 3000 transfection reagent. , 2018). 3. Transfection Transfection with Lipofectamine 3000 reagent and InvitrogenTM LipofectamineTM 2000 Transfection Reagent was compared. Lipofectamine™ Transfection Reagent is a trusted, value reagent with many years of cited use in thousands of publications and with many cell lines. Lipofectamine 3000 reagent demonstrates significant improvement in a broader spectrum of cell lines when compared to current commercially available lipid-mediated transfection reagents. HyCell™ CHO allows direct or sequential adaptation. Methods Figure 1. Available in 0. Transfection efficiency A375, CHO-K1, H1299 and HCT116 cell lines were transfected with a GFP expression construct using FuGENE® 4K or Lipofectamine™ 3000 (ThermoFisher Scientific) with varying reagent:DNA ratios. The most common sizes are listed below. According to the protocol Jun 1, 2004 · Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. 1 mL Explore Lipofectamine 3000 transfection reagent, offering high-efficiency transfection and improved cell viability in the broadest range of cell types. Transfection efficiency varies significantly among cell lines, with 74% efficiency for CHO-K1 using Turbofect. 1 Cell line-dependent differences in transfection efficiency. Invitrogen product line such as Lipofectamine 2000 reagent and Lipofectamine 3000 reagent were used to transfect 17 cell lines with a GFP-expressing plasmid in a 24-well plate format, using 0. I have recently tested Lipofectamine 2000 and a Cyan Fluorescent Protein (CFP) plasmid on CHO adherent cells. Background Lipofectamine 3000 is a new transfection reagent which is claimed to be more efficient than other transfection reagents like Turbofect. When I used the same reagent to transfect 293T cells I got around 90% of May 11, 2021 · Lipofectamine 3000 试剂是一种专门配制的温和转染试剂，经过优化可用于将质粒递送至 CHO 细胞，效率高且细胞毒性低。 该试剂可在存在或不存在血清的情况下进行转染。 CHO-S cells were transfected with pCMV•SPORT-βgal DNA (0. Lipofectamine 3000 reagent is a specially formulated gentle transfection reagent that is optimized for plasmid delivery into CHO cells with high efficiency and low cell toxicity. Cas9 protein and HPRT1 gRNA were transfected Lipofectamine 3000 reagent is a specially formulated gentle transfection reagent that is optimized for plasmid delivery into CHO cells with high efficiency and low cell toxicity. The Dec 5, 2022 · The single use of Lipofectamine 3000 represents a great advantage over the combinational use of Lipofectamine 3000 and P3000 by reduced toxicity. 5 μL of Lipofectamine 3000 reagent and 1 μg of DNA were each diluted in 50 μL GibcoTM Opti-MEMTM Reduced-Serum Medium; then 2 μL P3000TM reagent was added to the diluted DNA. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion Invitrogen Lipofectamine 3000 Transfection Reagent is our highest efficiency reagent for difficult-to-transfect cells. Keywords: Transfection, viral transduction, Lipofectamine 3000, EGFP, connexin43 Introduction Manipulation of gene expression holds much promise in the field of biomedical research. Sep 14, 2022 · From Thermo Fisher Scientific (Waltham, MA, USA), two reagents, Lipofectamine™3000 (LF3000) Transfection Reagent as a lipid nanoparticle-based technology, and Turbofect as a lipid-based cationic polymer transfection reagent were chosen. To simplify the transfection optimization process, we have created a transfection selection tool that provides recommended transfection reagent with optimized protocol for 120 cell lines. Lipofectamine 3000 and Lipofectamine RNAiMAX served as controls. Transfection should be done when dishes are 50-80% confluent. 16 μg to 0. Further optimization was performed to ensure reproducible and reliable results with the newly developed Lipofectamine 3000 reagent. This product offers versatility for DNA, RNA, and co-transfection use. While it is difficult to state the best Lipofectamine, since this highly depends on the experimental requirements, there are some general considerations. Lipofectamine 3000 reagent is a specially formulated gentle transfection reagent that is optimized for plasmid delivery into CHO cells with high efficiency and low cell toxicity. This chemically defined CHO medium is designed for large-scale culture applications, including perfusion and fed-batch feeding strategies. So, I have these two plasmids which I need to transfect say X and Y. kdylx lia rzrivae tkci pxyhe josys jxqlv amsonp rhnex cjnjsk wmla idyf pwyp aabyeow jlrgqy