Best transfection reagent for hek293 jetMESSENGER ® mRNA transfection reagent for hard-to-transfect cells. Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection If the viability of HEK293 cells being transfected is affected Feb 18, 2022 · Transfections allow for transient expression of a gene of interest in cell culture. Change the medium at 12~16 hours after TransfectNow™ HEK293 Cells performed equivalently to HEK293 cells from continuous culture, demonstrating both comparable transfection efficiency and assay window across 192 different kinase-NanoLuc® fusions (Figure 2). This procedure is suitable for CHO-S and HEK 293 GnTi- suspension cell Transient transfection of HEK 293 cells in a 75 cm3 flask (TFP08. Form: Liquid. 5 ml of Invitrogen™ Lipofectamine™ LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. 5 ml of complete growth medium 12-24 hours prior to transfection. Lipid-based reagents like Lipofectamine 3000, polymers such as PEI, and electroporation are all viable options, each with its advantages and limitations. This process may involve choosing a new transfection reagent. Transfecting HEK 293 Cells Use this procedure to transfect plasmid DNA into HEK 293 cells in a 24-well format (for other formats, see Scaling Up or Down Transfections, below). General Considerations 3 A. 2, Ref. The results from the study can have important implications on gene expression and cell line engineering experiments Passage the cells 18-24 hours before transfection to ensure the cells are actively dividing and that they will be at the appropriate cell density at the time of transfection. CRL-1573) using Lipofectamine LTX Reagent. The results showed that Transfection Reagent 2 had the best transfection e˜iciency with good viability among tested reagents, making it the top choice for transfecting HEK293 cells. 5 cm dish according to manufacturer’s instructions. The transfection is more efficient if the cells are in suspension. In order to address this issue and improve productivity, Polyplus-transfection ® has developed a novel powerful transfection reagent to generate superior protein amounts by TGE in HEK-293 and CHO 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. 0 x 10 6 cells/mL at time of transfection. Cationic Lipids 1 C. You can also tap the flask to loosen the cells. HEK293 cell transfection procedure: For transfection, add the expression vector plasmid DNA to the cells at a final concentration of 3 µg/ml and PEI to a final concentration of 9 µg/ml of transfection 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. Note: High serum levels (>5%) with antibiotics usually do not have inhibitory effect on transfection efficiency. Therefore, the DNA:PEI complex is endocytosed and the DNA is released into the cytoplasm. 293-Free<TMSYMBOL></TMSYMBOL> Transfection Reagent has a unique polycationic liposomal formulation and is designed expressly for transfection of HEK293 cells grown in suspension culture. High-efficiency, low-toxicity transfection of many cell types, including iPS cells. HEK-293 transfection optimization experiment. HEK-293 cells were cultured in DMEM media and Transfected with ptgs2. Transfection Add the mixture to the flask containing the HEK293T cells all at once and shake gently making cross movements to ensure good mixing. Therefore, efficiently deliver genes into HEK293 is very important. 5 µL of a transfection reagent (diluted in dH 2 O), and 7 µL of 0. I'm trying to lipid transfect HEK 293T cells using Qiagen's hiperfect transfection reagent. , 2018), or a defined N/P ratio, typically between 3:1 and ~25:1 (Ahn et al. My lab is looking for an alternate transfection reagent to Fugene 6 for transient transfection with minimal toxicity into HEK 293 cells. It is designed for the transfection of HEK293 cells grown in suspension culture and is ideal for mammalian protein production. Reagent Selection 3 Figure 3. 5×10 7 cells were Depending on the type of viral vector, different subtypes of HEK-293 cells are preferred such as HEK-293 for adenovirus and AAV virus production, HEK-293T and HEK-293FT for AAV and lentivirus production. 4. This reference provides a recommended procedure to transfect plasmid DNA into HEK 293, human embryonic kidney cells (ATCC No. Cell addition of Transfection Enhancer reagent. FuGENE® 6 Transfection Reagent. Chemical Reagents 1 B. The results showed that Transfection Reagent 2 had the best transfection efficiency with good viability among tested reagents, making it the top choice for transfecting HEK293 cells. Table 1 presents recommended starting conditions depending on culture vessel size. The transfection Aug 16, 2023 · High yields in suspension CHO & HEK-293. Advanced formulation of reagents and optimized transfection protocols provide efficient cellular delivery of protein, DNA, mRNA, shRNA and siRNA Add 1 ml of transfection mixture dropwise to each 10cm plate of HEK293 cells and gently shake the plate to distribute transfection reagent evenly. Sep 1, 2015 · High yields in suspension CHO & HEK-293. Then incubate for 24 hours in the presence of different Any time a new parameter, like a new cell line, is introduced, you will need to determine the optimal conditions for transfection. Asked 18th Sep, 2019; The aim of this research was to study the expression of vegf111b in HEK293 human cells. Plate 0. com In conclusion, the selection of the best tools and media for HEK293 cell transfection is a critical step in achieving successful gene delivery. Sep 17, 2021 · Specifically for HEK293 cells, transfect cells with 1 μg Myc-MG53 plasmid using 2 μL P3000™ Reagent and 3 μL Lipofectamine™ 3000 Reagent per 3. Jun 30, 2015 · Effects of different ratio of DNA and Lifectamine in transfection. SKU: MIR 2704 However, like in HEK293 cells, the protein expression in the BJ fibroblasts was lowest after the transfection with Stemfect RNA transfection reagent compared to other transfection reagents. Feb 20, 2022 · Optimized transfection is usually based on a defined mass ratio of transfection reagent to pDNA, typically between 1:1 and ~40:1 (Ahn et al. Find best transfection reagent for hek293 and related products for scientific research at Merck Protocol vA (June 2023) www. , 5–20 minutes at room temperature for the ViaFect™ Transfection Reagent). jetPEI ® Here, we describe a method for high throughput transient transfection of Human Embryonic Kidney 293 (HEK293) cells in 30 mL tubespins using polyethylenimine (PEI) as a transfection reagent. when choosing a transfection reagent for scale-up of transient transfection using HEK293 cells. The Reagent is ideal for transient transfection of HEK293 cells in large scale for production of recombinant proteins, monoclonal antibodies, virus particles (AAV, Adenovirus, Lentivirus) and virus-like particles. Physical Methods 2 D. Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection Optional: Transfection efficiency can be increased by addition of Transfection Enhancer reagent. doc Sep-01) page 1 of 1 User-Developed Protocol: Transient transfection of HEK 293 cells in 75 cm3 flasks using PolyFect® Transfection Reagent This procedure has been developed by customers for the transient transfection of HEK 293 cells Before Transfection. 13 Vector (Cat. Proven, dependable performance. Links. The complete FreeStyle 293 Expression Kit provides enough reagents to perform 16 transfections in a 30 ml volume, but larger volume transfections may be performed using simple scale-up of reagents. 26). Enhanced transient protein production in CHO cells. During development of each reagent, good transfection efficiency was verified in a variety of cells (as indicated in the product manual). This protocol describes how to transfect suspension HEK293 cells with recombinant antibody plasmids using Polyethylenimine Max as a transfection reagent. The recommended cell density for HEK 293 at transfection is ≥80% confluence. An automated liquid handler can be used to perform pipetting steps for transfecting batches of 96 tubespins, and septa in the tubespin caps allow for rapid Dec 6, 2020 · Use a Suitable Transfection Reagent:Select a transfection reagent that is compatible with large DNA plasmids and suitable for HEK293T cells. , 2008, Backliwal et al. TransIT®-293 Reagent Achieves High Transfection Efficiency. If the viability of HEK293 cells being transfected is affected at 16-24 hours post-transfection, changing the growth medium and eliminating redundant exposure of cells to transfectant can reduce the level of cytotoxicity. Sep 1, 2017 · For the selection of the best transfection reagent, 5 µL of cDNA (1 µg), 3 µL of 0. jetPRIME ® Versatile DNA/siRNA transfection reagent. Transfection Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection. polyplus-transfection. , 2018). Lipofectamine 2000_optimized resulted in best transfection efficiency with approximately 87% eGFP positive cells. Make sure that the cells are healthy and are ≥ 90% viable, prior to transfection. After transfection for 6 h, switch to fresh culture medium. jetOPTIMUS ® Best-in-class DNA transfection reagent. In order to achieve the best transfection efficiency, suggestions below yield high efficiency transfection in HEK 293 cells and derivative cell types using TransIT-293 Transfection Reagent. Which is the best transfection reagent for NIH3T3? Question. PEIpro ® is suitable for virus production in both adherent and suspension subtypes of HEK-293 cells. OriGene developed a transfection reagent, 293Tran, which was formulated specifically for HEK293 cells for high transfection efficiency. 4 M sucrose (for a final concentration of 64 mM) diluted in OptiMEM media, 3. HEK293 cells are also used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy. 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. HEK-293 Transfection Reagent Altogen Biosystems is a life sciences company that offers cell type-specific and optimized transfection products, electroporation kits, and in vivo delivery reagents. Fugene 6 works great, but is very expensive and poses a Specifically for HEK293 cells, transfect cells with 1 μg Myc-MG53 plasmid using 2 μL P3000™ Reagent and 3 μL Lipofectamine™ 3000 Reagent per 3. HEK-293 cells were grown to 85% confluency, then harvested and plated in a 96-well plate at 2 × 10 4 cells/100µl/well. Turbo293™ Transfection Reagent is specially optimized for efficient delivery of DNA into HEK293 cells. The day before transfection, trypsinize and count the cells. Fugene 6 works great, but is very expensive and poses a How do you transfect HEK293T cells? The protocol for a 24-well transfection reaction with HEK293 cells is here: Plate 10,000-15,000 HEK293 cells per well in 0. The optimal time for complex formation varies for each cell line. As a starting point, the amount of transfection reagent should be varied while keeping a constant plasmid DNA concentration (for example, 1:1, 3:1, and 5:1 ratios of volume to mass). Details: BOC Sciences HEK293-Transfection Reagent is a unique formulation based on polycationic liposomes of non-animal origin. Lipid-based transfection reagents such as Lipofectamine Transfection with lipofectamine to HEK293T shows a high efficiency even at very low dose of the reagent; we normally use 1-2 microgr/ml while for most monolayer cells the dose would be 4-5 microgr Sep 17, 2019 · However, the protein yields usually remain lower with TGE than with stable clones, making transfection a bottleneck in many bioproduction processes. However, the transfection success rate with calcium My lab is looking for an alternate transfection reagent to Fugene 6 for transient transfection with minimal toxicity into HEK 293 cells. , 2013, Fuenmayor et al. Best for everyday use with common cell lines. 15%) were combined to create a printing solution. Cells were detached as previously described and 1. 1, Fig. com FectoVIR®-LV is a novel class of animal free transfection reagent specifically developed for industrial scale production of lentiviral vectors (LVV) in suspension HEK-293 cell types. E2691, E2692, E2693. E4981, E4982. 4% gelatin (for a final concentration of 0. Step II. 25x105 cells per well in 0. Though the TransfectNow™ HEK293 Cells were optimized for use in NanoBRET™ Target Engagement assays, they may also be I. , 2008, Bollin et al. The results from the study can have important implications on gene expression and cell line engineering experiments as it helps to identify the most effective See full list on promega. Sep 1, 2021 · For ov erexpression of MG53 in HEK 293, transfect cells with plasmids using Lipofectamine 3000 Reagent following. 293Tran, which was formulated specifically for HEK293 cells for high transfection efficiency. Transfection. Transfection reagents need time to form the transfection reagent:DNA complex (e. After transfection and expression, the recombinant antibody can be purified for use in a variety of incubator while preparing DNA and PEI mixtures for transfection. , 2008, Solomun et al. Our laboratory prefers PEI to other cell transfection reagents because of its inexpensiveness. Including HEK293 transfection reagent and transfection enhancer. jetPEI ® How to transfect HEK293 suspensions samples with oligonucleotides or plasmids using polyethylenimine PEI STAR transfection reagent Keywords: polyethylenimine, pei transfection reagent, cationic polymer, HEK293 Suspensions, transfection reagent, nonviral transfection,pei polyethylenimine Created Date: 20221207140417Z Apr 6, 2024 · PEI compacts DNA into positively charged particles that adhere to anionic cell surfaces. Only use media that is reduced-serum, serum-free or chemically defined. , 2021, Zhang et Also note, HEK293 is a hard to transfect cell line as stated by a several transfection reagent booklets, it would be advisable to use transfection reagents like Lipofectamine 3000 (instead of 2000 I was wondering if someone could help with siRNA transfection. Identification HEK293 cells are also used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy. , 2011, Cervera et al. What is the best transfection reagent for HEK293? 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. • HEK293 cells, ~50,000 cells/cm2, seeded the day prior to transfection HEK293 cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal 80% confluency at the time of transfection (around 2x10^5 HEK293 cells). ViaFect™ Transfection Reagent. Seeding density. 0 x 10 6 cells/mL. See Choosing a transfection reagent is easier when you can rely on the work of researchers who have used it before you. and viability of several reagents in HEK293 cells. suggestions below yield high efficiency transfection in HEK 293 cells and derivative cell types using TransIT-293 Transfection Reagent. g. 1. Optimal transfection conditions for HEK-293 with the FuGENE® HD Reagent were determined using the protocol recommended in Figure 2. Cell density should be 50-80% confluent on the day of transfection. HEK 293 cells transfected with pEGFP using Trans IT®-293 Reagent in complete media for 24 hours. Invitrogen™ Lipofectamine™ LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. FectoCHO ® Expression System. 293Tran is a polymer-based and biodegradable transfection reagent. All amounts and volumes are given on a per well basis. FectoVIR®-LV transfection reagent guarantees higher LVV This approach can be adapted for different cell lines and different transfection reagents. Workflow Timeline Day 0: Seed HEK293T cells (or a subclone of HEK293T optimized for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection - Remove media, replace with fresh media Day 3 or more (am): Transfection and expression experiments may be performed directly in FreeStyle 293 Expression Medium without the need to change media. Chemical based The use of calcium phosphate as a transient transfection reagent is one of the most well-established and least expensive methods for adherent cell transfection at low volumes. 1 answer. # Transfection Reagent Materials • ™PEI STAR , 1 mg/mL, pH neutralized, sterile-filtered • HEK293 expression medium or DMEM maintained at 37°C transfection is inhibited by serum. Specialized for HEK293 – Maximize transfection performance in HEK293 cells; High Delivery Efficiency – Achieve 75-85% transfection efficiency; Low Cytotoxicity – Maintain cell Incubate DNA with transfection reagent for the optimal time. Immediately prior to transfection, ensure that viability is greater than 95% and viable cell density is 1. For example, one reagent may work well with HEK-293 cells, but another reagent may be a better choice when using HepG2 cells. 5 to 2. We would like to show you a description here but the site won’t allow us. Purchase HEK293 Transfection Reagent. HEK293 cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal 80% confluency at the time of transfection (around 2x10^5 HEK293 cells). 2). Subculture and expand cells to obtain culture with viability greater than 95% and viable cell density between 1. 5 -1. Materials The optimal ratio of transfection reagent to DNA is highly cell type-dependent. ProFection® Mammalian Transfection System. Cell density (% confluence) at transfection. The next day, the pGL4. Apr 1, 2024 · For transfection in static suspension, HEK293T cells were grown as a monolayer on adherent tissue culture grade plasticware as described above (Section 2. Reserve an aliquot of the culture for cell counting to confirm cell density. Viral Methods 3 II. Introduction 1 A. lyh mxglr xiy cfttwazb tnohmj wrqcu gkmb dchbrbd uai itukamv