Hek293 transfection reagent
Hek293 transfection reagent. Sep 9, 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. - Find MSDS or SDS, a COA, data sheets and more information. Fugene 6 works great, but is very expensive and poses a Jul 19, 2016 · Here, we use the transient expression of an A. thaliana guard cell outward-rectifying K + channel, AtGORK (At5G37500) in HEK-293 cells as an example to assess current commonly used transfection reagents and the fluorescent detection methods, and provide a specific protocol that is easily accessible for the general expression of membrane This is in part due to the fact that Expi293F Cells are adapted to achieve higher pg/cell/day productivity than standard HEK 293 cells, and the Expifectamine 293 Transfection Reagent and enhancers enable high-efficiency transfection and expression levels of high-density HEK 293 cultures. 25x105 cells per well in 0. Jul 22, 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. Unique Formulation – Maximize transfection performance in HEK 293 cells High Efficiency Delivery – Achieve 75-85% transfection efficiency to ensure experimental success Deliver Single or Multiple Plasmids – Ideal for specialized applications, such as virus production My lab is looking for an alternate transfection reagent to Fugene 6 for transient transfection with minimal toxicity into HEK 293 cells. The optimal time for complex formation varies for each cell line. Apr 21, 2021 · However, non-liposomal reagents were shown to produce higher transfection efficiencies than liposomal-based reagents in cell lines, including MCF-7, HepG2, 4T1, HCT116 and HEK293 (Yamano, Dai & Moursi, 2010; Homann et al. 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. Transfection reagents need time to form the transfection reagent:DNA complex (e. ATCC achieved transfection efficiencies of 99%, using the protocol described below. The transfection kit consists of the cationic lipid-based ExpiFectamine™ 293 Reagent and ExpiFectamine™ 293 Transfection Enhancers 1 and 2. It is designed for transient transfection of high density cultures of HEK (human embryonic kidney) 293 cells. 0 – 3. The enhancers are added 18 to 22 hours post-transfection to boost transfection, cell viability, and protein expression. PolyFect Transfection Reagent is stable for 1 year at 2–8°C. , 2011), Vero cells (Fliedl and Kaisermayer, 2011), CHO cells (Derouazi et al. HEK293 cells (or other cell line) at 70-90% confluency in a T-75 flask; EnGen ® Cas9 Nuclease NLS, S. FuGENE® 6 and FuGENE® HD Transfection Reagents resulted in the lowest decrease in viability for all three cell types tested with greater than 90% viability (Figures 2–4). Quality Control Endotoxin levels are ≤10 EU/ml determined using a Kinetic-QCL test (BioWhittaker, Inc. Advanced formulation of reagents and optimized transfection protocols provide efficient cellular delivery of protein, DNA, mRNA, shRNA and siRNA Invitrogen™ Lipofectamine™ LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. I am trying to transfect a GPCR into HEK-293 cells. See How to produce recombinant adeno-associated virus in adherent HEK293 cells using polyethylenimine PEI STAR transfection reagent Keywords: polyethylenimine, pei transfection reagent, adherent HEK293, transfection reagent, recombinant adeno-associated virus, rAAV, pei polyethylenimine Created Date: 20221214095244Z Apr 20, 2018 · Two days after transfection in HEK293 cells using Lipofectamine 3000 or three days after viral transduction in HUVECs with lentivirus generated from HEK293 cells utilizing Lipofectamine 3000 reagent, the cells were harvested, and GFP was clearly in these harvested HEK293 cells (A, tube 1) and HUVECs (B, tube 1) even without using a confocal Jul 24, 2017 · Hello, I have been having some issues with getting efficient transfection using PEI. You can also tap the flask to loosen the cells. 13 Vector (Cat. 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. Compared to calcium-mediated transfection, PEI is significantly more reliable. This reference provides a recommended procedure to transfect plasmid DNA into HEK 293, human embryonic kidney cells (ATCC No. Lipofectamine™ 2000 transfection reagent was the most toxic, and produced as high as a 55% drop in viability in HEK293 cells (Figure 2). Reserve an aliquot of the culture for cell counting to confirm cell density. Cell Incubate DNA with transfection reagent for the optimal time. Cells were then analyzed after 24 hours for transfection efficiency utilizing flow cytometry. The high-efficiency, cationic, lipid-based transfection reagent and transfection enhancers are designed to power the highest possible level of protein expression from Expi293F™ cells cultured in Expi293™ Expression Medium I was wondering if someone could help with siRNA transfection. Feb 20, 2022 · There is no consensus for the optimal N/P ratio, pDNA or transfection reagent concentrations in HEK 293 cells (Schlaeger and Christensen, 1999, Bollin et al. 5 -1. 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. 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. Plate 0. Sterility tests guarantee absence of any Invitrogen™ Lipofectamine™ LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. After transfection for 6 h, switch to fresh culture medium. I'm trying to lipid transfect HEK 293T cells using Qiagen's hiperfect transfection reagent. 2, Ref. , 2009) or others (Boussif et al. CRL-11268™) is a human epithelial adherent cell. The next day, the pGL4. 6-fold increase in the expression yield without causing any appreciable cytotoxicity. High Efficiency Delivery – Achieve 75-85% transfection efficiency to ensure experimental success. This process may involve choosing a new transfection reagent. No. Our protocol is to transfect when cells are ~50% confluent A549, COS7, HCT116, HEK293, and HeLa cells were seeded in 96-well plates according to users guide and subsequently transfected with FuGENE ® HD Reagent & GFP expressing plasmid at various FuGENE ® HD to Total DNA ratios. Invitrogen™ Lipofectamine™ LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. TransfeX™ Transfection of Plasmid DNA into HEK293T/17 Cells HEK293T/17 (ATCC® Cat. 1-GFP plasmid using X-tremeGENE™ 9 DNA Transfection Reagent. Experimental result: NIH-3T3 cells were successfully transfected with pcDNA3. incubator while preparing DNA and PEI mixtures for transfection. The recommended cell density for HEK 293 at transfection is ≥80% confluence. The day before transfection, trypsinize and count the cells. 5 X 10 4 cells/well. Animal-free polycationic liposomal transfection reagent optimized for the transfection of HEK293 cells grown in suspension culture. Optimal transfection conditions for HEK-293 with the FuGENE® HD Reagent were determined using the protocol recommended in Figure 2. HEK-293 transfection optimization experiment. Any time a new parameter, like a new cell line, is introduced, you will need to determine the optimal conditions for transfection. 0 x 10 6 cells/mL. However, the transfection success rate with calcium Invitrogen™ Lipofectamine™ LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. Deliver Single or Multiple Plasmids – Ideal for specialized applications, such as virus production. Cell density should be 50-80% confluent on the day of transfection. transfection efficiency of plasmid DNA in HEK 293 and cell types of associated lineage. Lipofectamine 3000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection. Polyethylenimine (PEI) transfection is one of the most widely used methods to co-transfect plasmids into HEK293 cells. Unprecedented protein yields were reached in both CHO and HEK-293 cells, hence FectoPRO ® is the reagent of choice to generate high quality protein yields. Before Transfection. 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 Invitrogen Lipofectamine Stem Transfection Reagent is designed to efficiently deliver DNA, RNA, or Cas9 protein complexes into stem cells which are typically hard to transfect via reagent-based transfection methods. Plate HEK-293 cells at a density of 3. TransIT-293 provides all the attributes of the trusted TransIT series of transfection reagents: high transfection efficiency, low toxicity, serum compatibility, simplicity of use and reproducibility. CRL-1573) using Lipofectamine LTX Reagent. ExpiFectamine ™ 293 Reagent has the following features: • Designed specifically for transfection of high-density suspension cell culture, with matching transfection enhancers that boost transfection performance and protein expression HEK293 (and HEK 293-T cells which are easier to transfect) behave like this normally when they are overconfluent. , 2004, Bollin et al. General Considerations for using the TransfeX transfection reagent: Unique Formulation – Maximize transfection performance in HEK 293 cells. FectoPRO® Transfection reagent is specifically designed for enhanced Transient Gene Expression (TGE) in suspension CHO and HEK-293 cells in various serum-free media, using low DNA amount (< 1 µg/ml of cell culture). Note: High serum levels (>5%) with antibiotics usually do not have inhibitory effect on transfection efficiency. , 2017). , 2011, Ye et al. suggestions below yield high efficiency transfection in HEK 293 cells and derivative cell types using TransIT-293 Transfection Reagent. 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 HEK293 cells are also used by the biotechnology industry to produce therapeutic proteins and viruses for gene therapy. . 5 ml of Jun 18, 2013 · The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Links. 293 Reagent is optimized for the transfection of plasmid DNA into high-density Expi293 ™ cell cultures. All amounts and volumes are given on a per well basis. Jul 26, 2016 · This is a ‘reverse transfection’ method that uses a final concentration of 10 nM RNP per transfection in a 96-well culture plate. Many cell lines can be transfected successfully with PEI but in our experience these two cell lines express the highest level of protein compared to other cells. Table 1 presents recommended starting conditions depending on culture vessel size. 1, Fig. For example, one reagent may work well with HEK-293 cells, but another reagent may be a better choice when using HepG2 cells. 5 cm dish according to manufacturer’s instructions. Make sure that the cells are healthy and are ≥ 90% viable, prior to transfection. thaliana guard cell outward-rectifying K + channel, AtGORK (At5G37500) in HEK-293 cells as an example to assess current commonly used transfection reagents and the fluorescent detection methods, and provide a specific protocol that is easily accessible for the general expression of membrane 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. The transfection is more efficient if the cells are in suspension. Optimized, easy-to-use siRNA transfection reagent formulated for minimal toxicity. 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. 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). Figure 3. Lipofectamine Stem reagent is up to 3x more efficient than existing transfection reagents in iPSCs, NSCs, and MSCs. , 2004, Tait et al. 1. 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. 26). Required Materials: Cell Culture and Transfection. In this study, scientists from Aragen’s Discovery Biology (Reagent Generation) compared the transfection efficiency and viability of several reagents in HEK293 cells. Cell density (% confluence) at transfection. pyogenes (M0646T or M0646M) Invitrogen™ Lipofectamine™ LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. Sep 17, 2019 · ProteoGenix compared FectoPRO ® to other transfection reagents commercially available and concluded that FectoPRO ® clearly outperforms both PEI- and lipid- based reagents. Step II. Depending on the transfection reagent you should aim to transfect the cells Optimizes transfection performance in HEK 293 cells; Low cellular toxicity maintains cell density and reduces experimental bias; Allows 75 to 85% transfection efficiency in HEK 293 cell lines; Delivers single or multiple plasmid for specialized applications such as virus production; Supplied as a ready-to-use solution; Concentration: 1. Jul 18, 2016 · Here, we use the transient expression of an A. • 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). Subculture and expand cells to obtain culture with viability greater than 95% and viable cell density between 1. Results Turbo293™ Transfection Reagent is specially optimized for efficient delivery of DNA into HEK293 cells. 293-Free™ Transfection Reagent Animal-free polycationic liposomal transfection reagent optimized for the transfection of HEK293 cells grown in suspension culture. 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. Our laboratory uses PEI over other cell transfection reagents because of its low cost. Each reagent was used to transfect HEK 293, HeLa, LNCaP, HepG2, and A549 cell lines in a 96-well format, and GFP expression was analyzed 48 hours post-transfection. Transfection of HEK-293 Cells. Transfection: Transient and stable transfections Conditions: Works in the presence of serum, with no need to wash cells after transfection Shelf life: Long shelf-life of plasmid DNA (pDNA)/ LyoVec™ complexes Quality Control: Transfection efficiency of LyoVec™ has been confirmed in HEK293‑derived cells. This protocol is appropriate for two suspension cell lines, CHO-S and HEK 293 GnTi-. 41 mg/mL 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. Only use media that is reduced-serum, serum-free or chemically defined. # Transfection and expression experiments may be performed directly in FreeStyle 293 Expression Medium without the need to change media. 0 x 10 6 cells/mL at time of transfection. , 5–20 minutes at room temperature for the ViaFect™ Transfection Reagent). PEI STAR™ has been used to Add the mixture to the flask containing the HEK293T cells all at once and shake gently making cross movements to ensure good mixing. And, compared to lipid-mediated transfection, PEI is more scalable and cost-effective. Feb 2, 2021 · Results: By expressing green fluorescent protein (GFP) as a reporter protein using the calcium phosphate transfection method and imaging a large population of cells, we found that a 5-min exposure of 10 % DMSO to HEK-293 cells, 4 h after transfection of the protein of interest, leads to ~1. Our HEK 293 derived viral production cells have been optimized for viral production in suspension culture. Important guidelines for transfection. Transfection. , 1995, Ogris et al when choosing a transfection reagent for scale-up of transient transfection using HEK293 cells. HEK-293 cells were grown to 85% confluency, then harvested and plated in a 96-well plate at 2 × 10 4 cells/100µl/well. Lipofectamine 3000 reagent provided higher GFP transfection efficiency than Lipofectamine 2000 and FuGENE HD reagents for all five cell lines. 5 to 2. Purchase HEK293 Transfection Reagent. 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. The recommended cell density for HEK 293 at transfection is 50–70% confluence. Optimized transfection reagent specifically developed for mammalian protein production in chinese hamster ovary (CHO) cells. Immediately prior to transfection, ensure that viability is greater than 95% and viable cell density is 1. Transfection The Gibco LV-MAX Lentiviral Production System includes: HEK 293–derived suspension cells, serum-free media, proprietary transfection reagent, supplement, and our novel enhancer. Seeding density. 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. g. ) PolyFect Transfection Reagent is tested by transfection of pCMV β(CLONTECH) into HeLa-S3 and COS-7 cells to ensure lot-to-lot consistency. agnddav xwrm heq efmxr chwlit ccikr axxw bwxm jngmvc surkuh