expi293f transfection protocol Recombinant MxB was transiently expressed in mammalian cells using the Expi293 Expression kit from Life Technologies (Invitrogen). Binding kinetics were determined using biolayer interferometry with an Octet Red instrument. The cells are then collected by centrifugation and resuspended At 16 hr post‐transfection, 1. First, pre-clear your samples of cells and cellular debris, and then simply add the appropriate amount of ExoQuick-TC to your cleared biofluid, refrigerate, and centrifuge (see the product manual for protocol details). As for all transfection methods, electroporation has its advantages and disadvantages. For metabolic (methionine) protein labeling, Expi293 ™ Met (-) Expression Medium and the ExpiFectamine ™ 293 Met (-) Transfection Kit should be used to metabolically label The Expi293 Expression System provides rapid, high-yield protein production in mammalian cells by combining high-density Expi293F cells, an optimized medium, specialized transfection reagent and enhancer. The TransIT-PRO ® Transfection Reagent & Kit uses animal origin free components designed for high and reproducible protein yield in suspension CHO and 293 derived cells. , more efficient production) and indirect aspects (i. Rigau M et al expressed BTN2A1 antibody clones in Expi293F cells [ 10 ]. The S protein and RBD antigens were expressed and purified from pCAGGS-RBD and pCAGGS-solSpike vectors respectively, kindly provided by Florian Krammer (The Icahn School of Medicine at Mt Sinai, New York City, NY, USA) using Expi293F or Freestyle293 human embryonic kidney (HEK) cells and published protocols (Stadlbauer et al. Protein expression overview Recombinant protein expression technology enables analysis of gene regulation and protein structure and function. Prep time 15 min, run time 2 min. We recommend adding the Thromboplastin D protocol before adding the ExoQuick, per the steps in the user manual. Expi293F cells at 2 × 10 6 /ml were transfected with a mixture of 1 ng of coding plasmid (i. Lipofectamine® transfection reagents are the most trusted and cited The plasmid was transfected into Expi293F cells by using ExpiFectamine 293 (Thermo Fisher Scientific) according to the manufacturer’s protocol. Expi293’s ease of use, flexibility, and efficiencies provide researchers a straightforward, cost effective method for improving protein production. 9. 4-TOPO ® (30 μg) was used to transfect 7. Our mission is to enable our customers to make the world healthier, cleaner and safer. CHO cultures were fed every 2 days with 5% v/v EfficientFeed B. As a result, the transfection procedure is identical to the transfection of Freestyle™ 293-F cells with the exception that cells are propagated and maintained in the 50:50 mix of Ex-Cell/Freestyle media until the time of transfection. e. 5–5 μg/μL) 30 μg Opti-MEM™ Medium 1. Plasmid DNA was added at 3. Cell Selection Post-Transfection. Different transfection protocols were compared: A) Commercial Lipid base reagent, B) K2 transfection system, C) 25kDa Linear PEI with Freestyle medium , D) 25kDa Linear PEI with SFM4Transfex medium and E) Negative control (untransfected cells) The Expi293 Expression System is a major advance in transient expression technology for rapid and ultra high yield protein production from mammalian cells It is based on high density culture of Expi293F cells in Expi293 Expression Medium Transient expression is powered by the cationic lipid based ExpiFectamine 293 transfection reagent in combination with specialized transfection enhancers All components work in concert to generate 2 to 10 fold higher protein yields than previous generation Expi293F™ cells are usually transfected at higher density and for transfections with FectoPRO you can either follow our protocol and seed the cells at 1. This product (s) resides on a Fisher Scientific GSA or VA contract. Use of TransIT-PRO Transfection Kit eliminates the need for a culture medium change post-transfection and is suitable for both transient and stable expression. 25 μg, respectively), and due to several media exchanges, the use of 10-stack Cell Factory systems used 3-fold more media than did SJ293TS cells in 5-L shaker flasks. Culture media were harvested three days post-transfection by centrifugation. In our case Expi293F cell media (~ 123. Expi293f cells (2. , more efficient downstream usage) of antibody development pathways. Thus, the non-engineered Expi293 F cell line, which had been selected for its transfection efficiency, productivity, and growth rate , was used as the host cell line for optimization of the transfection procedures. Culture conditions and transfection of HEK293F suspension cells. Note:"Customer transfection protocols"に記載されている実験条件および実験結果等に関する記載は、データを提供されたX-tremeGENEユーザに帰責します。 X-tremeGENE HP DNA Transfection Reagent As in the case of mouse Wnt3a, 12 human Wnt proteins exhibited no or very little secretion into the serum-free culture media when singly transfected into the Expi293F cells (Figure 7B, lanes marked with –). Subsequently, the photocage is removed with In this article, we report the development of a novel transient transfection system—Expi293™—that utilizes high-density 293F cell cultures to generate expression levels greater than 1 g/L for human Multiple expression systems, vectors, and transfection reagents are available to prepare different formats of antibodies in vitro. Gxpress 293 Transfection & Medium Kit has been developed for the gene expression system of human embryonic kidney (HEK) 293 cells, such as Expi293F or 293T cells, and the high production of recombinant proteins or the transfection assay in serum-free culture. Transfection Enhancers were added 18 h post-transfection, and cells were cultured for six to seven days. The inoculum containing the rHSV stocks (rHSV-GFP [UF5], and rHSV It is designed for transient transfection of high density cultures of HEK (human embryonic kidney) 293 cells. 5 mL suspension was added per well. Gxpress 293 Transfection Kit For 293 cells "Expi293F, 293-F, HEK293, 293T, etc. • High FiveŽ cells double every 18-24 hours. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously. 5 versus 506. As with many • Implemented polymer based transfection protocols for siRNA and evaluated RNA interference-mediated gene silencing (VEGF and HIF1A). The complexation reaction mixture was further incubated Transfection Reagent Lipofectamine® MessengerMAX™ Transfection Reagent ExpiFectamine™ 293 Transfection Kit Recommended for Expi293F ™ cells Broad-spectrum transfection reagent Lipofectamine® 2000 Transfection Reagent Electroporation Neon® Transfection System In vivo delivery The Trans I T-PRO® transfection kit from Mirus Bio was specifically developed for high yield mammalian protein production in suspension HEK 293 and CHO derived cells. Protein supernatant was harvested on day 5/6 after transfection, spun, and 0. HEK Expi293F cells and CHO‐S cells were transfected with plasmids encoding spike gene using PEI under optimized conditions and cultured at 37°C or 32°C. HiSpeed Maxiprep Kit) were performed using the Expi293 Expression System from ThermoFisher (Catalog # 14524, Expi293F cells, Expi293 Media, and the ExpiFectamine 293 Transfection Kit). The inoculum containing the rHSV stocks (rHSV-UF5 (GFP), and rHSV-AAV9) in appropriate amounts was prepared in the growth medium and directly added to the cell suspension culture. The Expi293 system is the latest evolution of transient transfection technology in an easy to use and complete system. , library DNA) with 1. pointed out that exosomes, as an ideal drug delivery tool, require cargo that causes minimal adverse effects and should have The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. OptSpike2 in Expi293F and ExpiCHO-S expression systems20 and provide detailed protein production protocols, including a Standard Operating Procedure (SOP) in the Supporting Information Appendix 1. TransIT-PRO® transfection kit and reagents are ideal for biotherapeutic protein production in suspension CHO and 293 cells. We then evaluate the biochemical and biophysical properties of these proteins to define standards This pair, in response to a nonsense codon (UAG), charges a photocaged-Cit, SM60 (Fig. 5 μg of pCEP4-ΔCMV carrier plasmid [described in ]. The CH129 mAb was obtained following transient transfection of Expi293F™ cells using the ExpiFectamine™ 293 Transfection kit (Gibco, LifeTechnologies). Polyplus-transfection ® has a strong expertise in developing GMP grade transfection reagent, recently marked by the launch of the first GMP compliant transfection reagent for therapeutic virus production (PEIpro ®-GMP in 2018). 1b, c), into proteins expressed in HEK293T or EXPI293F cells. The canine PD-L1-Fc and its mutants in addition to the cynomolgus, murine and rat PD-L1-Fc were generated in a manner similar to that of the human PD-L1-Fc. Hybridomas were further expanded and antibodies were purified. On the plus side, you can use it to transfect large DNA fragments and achieve good transfection efficiencies using cell lines. 5 × 10 7 EXPI293F™ suspension cells (2. Since it is compatible with varied media formulations, the same media can be used for both transient and stable expression. 4-IGG-PGLYRP. 8 µg DNA/mL), ExpiFectamine™293 + Enhancers (1 µg DNA/mL), or FreeStyle™MAX (1. Combining a high efficiency, reproducible, animal origin-free DNA transfection reagent (TransIT-PRO) and an optional boost reagent that enhances expression in certain media formulations (PRO boost), the TransIT-PRO This protocol for the production of rAAV9 in suspension was built upon the one developed for adherent HEK293 with the following changes: EXPI293F™ cells were counted and homogenized in fresh medium at a concentration of 1×10 6 cell/mL on the day of infection. The purified protein was used as an immunogen for production of monoclonal antibodies in mice. In additiontoGFP-LV,amorecomplexLVproduction,V5m3-Sardinia We currently offer recombinant protein production in human (Freestyle 293-F and Expi293F) and insect (Sf9 and High Five) cell lines using transient transfection. The TransIT-PRO® Transfection reagent and kit use animal origin free components designed for high and reproducible protein yield in suspension CHO and 293 derived cells. Automation of transient transfection begins with manual experiments to determine the optimal ratio of DNA to lipid, and the optimal ratio of the DNA:lipid complex to cells. 3 pcDNA™3. Expi293F cells were transfected using an Expifectamine 293 Transfection kit (Gibco, Carlsbad, CA), following the manufacturer's protocol. Transfection and expression experiments may be performed directly in Expi293™ Expression Medium without the need for media change. For Protocol for Transfecting Expi293F™ Cells using Trans IT-PRO® Reagent Determine the total number of cells required for your experiment at least one day prior to transfection. As a next step, cells were cultured and selected in the presence of appropriate drugs. com/support. Figure 1: Efficiency of transfection of HEK293 cells using a reporter GFP assessed by FACS analysis. High protein yield in suspension CHO and 293 cells, including CHO-S and Expi293F™ cells She has extensive experiences in mammalian cell culture, serum and protein free, chemical defined medium formation as well as protein expression. However, transfection efficiency in primary cells is low, mostly due to the high mortality rates caused by the electric pulses. Doubling Time Population doubling times for insect cells will vary depending on growth conditions. Expi293F is the most commonly used suspension HEK293 cell line for transient expression to achieve high cell density. TransIT-PRO® Transfection Reagent & Kit . 5 × 107 cells Expi293™ Expression Medium 25. The inoculum containing the rHSV stocks (rHSV-GFP [UF5], and rHSV To establish stable cell lines, transfection is carried out in the same way for transient expression, and the cells are grown without a selection drug for a few days. We have further optimized the protocol for IgG reformatting since the initial publication of this method (Chen et al. 22 µm filtered. 0 μg/ml PMab-1 in DMEM/FCS for 2 h on ice. transfection protocols, and can be used in metabolic protein labeling studies. " Gxpress 293 Transfection Kit has been developed for the gene expression system of human embryonic kidney (HEK) 293 cells, such as Expi293F or HEK293 cells, and the high production of recombinant proteins or the transfection assay in serum-free culture. It has broad applications across various fields of cell and molecular biology - wherever you want to measure or track expression of a cloned gene. Results The protocol has been used to generate multiple variants of tetra- and hexavalent bispecific inhibits DNA/PEI transfection). The ExpiFectamine 293 Transfection Kit is a core component of the Expi293 Expression System It is designed for transient transfection of high density cultures of HEK human embryonic kidney 293 cells 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 The protocol for use of ExpiFectamine™ 293 Transfection components is very similar to transfection protocols for other transient expression reagents, such as 293fectin™ and FreeStyle™ MAX. The plasmids for heavy and light chains for IgGs and Fabs were isolated using Qiagen maxi prep kits and cotransfected into Expi293F cells using the Expi293F Transfection kit (ThermoFisher Scientific) following the supplier’s protocol. 8 mg total DNA/liter cells) with FectoPro transfection reagent (Polyplus) at 1:1 DNA/FectoPro ratio Typically, this happens if starting with plasma or serum. 4 Max. 25 µg/mL) following the standard protocols. Although an earlier report suggested that the cells contained Adenovirus 5 DNA from both the right and left ends of the viral genome, it is now clear that only left end sequences are present. 4 €/L) calculated by the list prices without considering the costs for Glutamine Transfection is the process by which nucleic acids are introduced into eukaryotic cells. We have further optimized the protocol for IgG reformatting since the initial publication of this method (Chen et al. ExoQuick-TC is a proprietary polymer that gently precipitates exosomes. For the Expi293F™ cells, shake flask cultures at L scale were used1 , and transient transfection was performed as described in the manufacturer instructions. 1). 4 €/L) is ~ 35% more expensive than our High Five cell media (90. 6 /ml) were transfected with 100ng DNA and conditioned medium harvested at day seven post-transfection. 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 2 vials frozen Expi293F cells 1 bottle 1000 mL Expi293 Expression Medium 1 ExpiFectamine 293 transfection kit for 1 L of culture 1 vial Antibody Expressing Positive Control Vector 1 bottle 100 mL Opti-MEM Reduced Serum Medium Store all components at +2°C to +8°C, except frozen cells are stored in liquid nitrogen: Expression System: Mammalian 1 Protein expression handbook For educational purposes only. pcDNA™ 3. Per ml of 2 × 10 6 Expi293F cells, 500 ng of pcDNA3-sPDGFRα-Fc (IgG1) and 3 μg of polyethylenimine (MW 25,000; Polysciences) were mixed in 100 μl of OptiMEM (Gibco) and incubated for 20 minutes at room temperature prior to adding to cells. Briefly, HEK293 cells in suspension (100ml, 2x10. The recombinant virus titer was sequentially amplified from P1 to P3 virus stocks for protein production in the Sf9 insect cells and EXPI293F Expi293F cells (Life Technologies Corporation, Carlsbad, CA) were cultured in Expi293 Expression Medium (Life Technologies Corporation, Grand Island, NY). 8. The purified protein was used as an immunogen for production of monoclonal antibodies in mice. These studies could provide a mechanistic justification for novel targeted therapies in cancer. 5 mL DNA (0. Nucleic Acids Res 42:e26, 2014) and also updated the transient transfection protocol using Expi293F cells, which are described herein. A third protocols describes selection of transfected cells from the total population soon after transfection with plasmids that express both the gene of interest and a selection tag. 2 x 10 6 cells/mL in various culture media and cell density and viability were This protocol for the production of rAAV9 in suspension was built on the one previously developed for adherent HEK293 cells with the following changes: Expi293F cells were counted and homogenized in fresh medium at a concentration of 1 × 10 6 cells/ml on the day of infection. Full methods are detailed in SI Appendix, Materials and Methods. 5 μg plasmid DNA. • Performed in vitro gene delivery system evaluation in Protein was expressed in Expi293F TM cells using the manufacturer’s protocol (Thermo Fisher). Expression levels of IgG 1 and Cytokine 1 were 70- and 57-fold higher in the ExpiCHO system than in the FreeStyle MAX CHO-S system, respectively. expensive media, cost of transfection reagents and higher capital investment). 0 ml of Expifectamine 293 Transfection Enhancer 2 from the kit are added to the culture, and subsequently the culture is returned to the shaking incubator. They were then purified from the culture medium and separated from the light chain of IgG by proteolysis . At 3 days post‐transfection, the cells are harvested and centrifuged 20 min at 4000 × g, 4°C. Expi293F cells were transiently transfected with each plasmid. • Performed in vitro gene delivery system evaluation in - Improved large scale transient production protocols using Expi293F human cells in 2L single use bioreactors and 10L bioreactors. . Protocol overview and potential applications Plasmids encoding IgG 1 and Cytokine 1 were transfected into FreeStyle CHO-S, Expi293F, and ExpiCHO-S cells and expression was determined 7, 5, and 11 days post-transfection, respectively (Fig. Theculturemediumwasdilutedin8 chromatographic buffer (final concentrations of 500 mM NaCl, 20 mM NaH 2 PO 4 To produce recombinant HIV mAbs for assay and controls, Expi293F cells were transfected following the manufacturer’s protocol for Expifectamine (Thermo Fisher). 5 × 10 6 cells/mL in 30 mL) in a 125 mL erlenmeyer, using 81 µL of ExpiFectamine Transfection, Expression, and Purification of the IgGs and Fabs. Also, transfection of HEK293T cells consumed 2-fold more plasmid DNA than did SJ293TS cell transfection (1,012. Viral supernatants were collected 48–72 h after transfection and in some cases concentrated using the Lenti-X TM Concentration kit (Clontech, Takara Bio) per the manufacturer's protocol. The recombinant fusion proteins were detected in the culture medium of Expi293F cells transfected with the pcDNA3. e. Li et al. 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 The second protocol includes conditions for thirteen markers commonly used for selection of mammalian cells. Antibody-Expressing Positive Control Vector is provided for transfection and expression in Expi293F ™ cells. For this purpose, the plasmid for transfection must contain a drug-resistant gene. 5 mL The protocol for use of ExpiFectamine™ 293 Transfection components is very similar to transfection protocols for other transient expression reagents, such as 293fectin™ and FreeStyle™ MAX. The bulk from cultures (55 L), per Antibody variants were expressed by transient transfection of Expi293F HEK cells and purified from supernatant by using protein A. 5 mL Opti-MEM™ Medium 1. Not only is Polyplus-transfection the provider of the first GMP transfection reagent for the Gene and Cell therapy All suspension cells from Invitrogen either 293F, Expi293F or ExpiCHO grow well in their respective culture medium with the following conditions: 37 degrees Celsius temperature, shaking 100 RPM, 8 High Efficiency Transfection of CHO-S Cells Using the TransIT-PRO® Transfection Reagent. Transient transfection protocol for HEK293T cells Protocol created by Lorena Maestre - Monoclonal Antibodies Unit, Centro Nacional de Investigaciones Oncológicas. Cells Life Technologies is now part of Thermo Fisher Scientific. Cell culture and transfection services are subsidized by our COBRE program (NIH IDeA award for bioMT P20-GM113132). The medium was replaced 2 hours after transfection, and cells were collected 24 hours after transfection for FACS. 5 ml) were transiently transfected with 10 μg of pBK-β 2 AR WT or pBK-β 2 AR Y219A and the ExpiFectamine 293 transfection reagent (Gibco). Every lot of EndoFectin™ Expi293 is tested by transfecting Expi293F cells with an eGFP-expressing plasmid (GeneCopoeiaCatalogNumberEX-EGFP-M02). ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific, A29129) OptiPRO Serum-Free Medium (Gibco, Thermo Fisher Scientific, 12309-050) 1 L non-baffled, vented flask, sterile (Fisher, PBV1000) Expi293F cells were transfected with mock or GFP plasmid. Prep time 15 min, run time 2 min. In the past five years, she served as leading scientist to focus on the development of two novel transient transfection systems, Expi293™ and ExpiCHO Expression Systems. For detailed protocols, refer to the Expi293™ Expression System Kit User Guide, available online at thermofisher. High density cultures of Expi293F™ cells that result in more expressing cells per milliliter of culture and higher protein yields; Native folding and mammalian post-translational modifications, such as glycosylation, of expressed proteins; Easy, robust culture and transfection protocols that readily fit into current transient expression workflows It is designed for transient transfection of high density cultures of HEK (human embryonic kidney) 293 cells. 0 at μg/mL for 24 h, after protocol to incorporate full-length RON and EGFR receptors into nanodiscs, as well as developing techniques to detect protein incorporation into these nanodiscs, which would ultimately facilitate structural studies for RON and EGFR heterodimerization. Use suspension cells directly or trypsinize adherent cells for analysis. 293 or Expi293F cells appear to be a popular choice [ 6 ]. GeneXPlus works effectively in large scale reactions, producing high protein yields. Transfected cells are important in investigating the specificity of antibodies and also permit studies on the regulation and function of proteins. EndoFectin™ Expi293 is a powerful new transfection reagent ideally suited for transfection of the suspension cell line Expi293F. To express mouse MCPH1, Expi293F cells (Thermo Fisher; maintained in Expi293 Expression Medium with an orbital shaker, 8% CO 2, 37°C) were cultured to 2. A complete list of cell line-specific protocols can be found here. We help our customers accelerate life sciences research, solve complex … Continued The AbVec vectors containing the heavy and light chain sequences of CR9114 mAb were kindly provided by the laboratory of Dr. However, their expression was enhanced upon the co-transfection with human AFM (Figure 7B, lanes marked with +). Nucleic Acids Res 42:e26, 2014) and also updated the transient transfection protocol using Expi293F cells, which are described herein. 5 × 10 7 cells in 25. For each antibody, plasmids encoding the heavy and light chain were transfected at a 1:2 molar ratio (0. Since it is compatible with varied media formulations, the same media can be used for both transient and stable expression. 106 cells/mL one day before transfection or seed the cells as recommended in the Expi293™ protocol. You can even get specific HEK293 transfection kits with all the reagents you need included to make it super easy. 8 × 10 6 cells/mL and then transiently transfected. ScreenFect™ UP-293 Booster, which is added to transfected cells, enhances remarkably protein expression level in cells. His-tagged codon optimized genes for mammalian expression were used for transient transfection of CoV-2 RBD and S protein ectodomain in Expi293F cells to produce the recombinant proteins and purified by Ni–NTA affinity chromatography following standard protocol based suspension celllinescommercially available, namely, Expi293F cells and Viral Production Cells. Over95%ofcellsexpressedeGFP16hourspost-transfection. Patrick Wilson (University of Chicago) and used for transient transfections of Expi293F cells using ExpiFectamin ™ transfection kit (Gibco) according to the manufacturer’s instructions. Since it is compatible with varied media formulations, the same media can be used for both transient and stable expression. Cells Expi293F ExpiCHO-S Media Expi293 Medium ExpiCHO Medium Transfection Reagent ExpiFectamine 293 ExpiFectamine CHO Control vector Antibody-expressing vector Expression Vector 2 pcDNA ™3. The rabbit IgG that is produced in Expi293F cells after transfection with the control vector is secreted into the Expi293 ™ Expression Medium, with optimal yields occurring 5–7 days post-transfection. 5 ml of Expi293™ Expression Medium. ScreenFect™UP-293 is a transfection reagent kit which is highly efficient for production of antibodies and proteins in Expi293F™ or FreeStyle™ 293-F cells. Because they grow quite rapidly, you can passage your HEK293 cells every couple of days (in our lab, we typically passage on Mondays, Wednesdays and Fridays). The protocols outlined here are thereby likely to improve both direct (i. Comments This reagent affords high levels of gene expression in a variety of cell types and is suitable for both transient and stable transfection. Expi293F or BHK cells were coinfected at the indicated MOI with rHSV stocks containing a gene of interest (encoding green fluo-rescent protein [GFP] or GAA) and a second rHSV containing AAV Rep2Cap2, Rep2Cap5, or Rep2-Cap9. 0 × 10 6 viable cells/ml prior to transfection. Cells were grown in shaker flasks at 37 C, 80% humidity, and 100 and 125rpm for BHK and Expi293F cells, respectively. Briefly, cells were diluted to a density of 3 × 10 6 cell ml −1 with a fresh medium. 2 ml of Expifectamine 293 Transfection Enhancer 1 and 12. Suspension-adapted Expi293F cells were grown in Expi293 Expression Canine PD-L1-Fc and its mutants were expressed transiently in Expi293F cells following transfection using ExpiFetamine 293. Transfection enhancers were added 18 hours after transfection, and supernatants were harvested 6 days after transfection. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated. into Expi293F expression system (Thermo Scientific) following the manufacturer’s protocol to produce recombinant mAbs. Forty-eight hours after transfection, 2. Hybridomas were further expanded and antibodies were purified. Target cells were seeded in growth media in a 6-well plate (Corning) and 1 ml virus was added with Polybrene at a final concentration of 10 μg ml −1 . The human embryonic kidney (HEK) cell line (HEK293T) has been used EVs donor for a broad range of investigations, due to their high transfection efficiency, ease of growth, and capacity for high yield of EVs . Cells were harvested after 48 h of transfection and incubated with the anti-FLAG M2 antibody (Sigma-Aldrich, F3165) diluted at 1:500 in Dulbecco’s Modified Eagle Medium (DMEM) with 10% foetal calf serum (FCS) or 1. Each 30 ml transfection requires 7. Since the reagent is composed of animal-origin Briefly, on the day of transfection Expi293F cell cultures were diluted to 3 × 10 6 cells/mL and 2. ATCC CRL-1573 (HEK293) cells were progressively adapted from an anchorage-dependent growth mode to a suspension mode. . This protocol for the production of rAAV9 in suspension was built on the one previously developed for adherent HEK293 cells with the following changes: Expi293F cells were counted and homogenized in fresh medium at a concentration of 1 × 10 6 cells/ml on the day of infection. 10ul of each sample was stained with ChemoMetec solutions and run on the NucleoCounter ® NC-3000™. EXPI293F cultures with >95% viability were centrifuged and resuspended in fresh medium at 3 × 10 6 cells/mL within 1 h prior to transfection. Plasmid DNA encoding EGFP was delivered by transient transfection with the TransIT-PRO® Transfection Reagent (1:1) (reagent:DNA ratio, volume:weight) using 1 µg plasmid DNA per milliliter of culture and cell a density of 2 x 10 6 cells/ml in the CHOgro® Expression Medium at the time of transfection. , 2020), but with a modified transient transfection protocol using polyethyleneimine (PEI). A master polyplex was formed, and an equal amount was distributed to each flask. Expi293F cells maintained in Expi293 expression medium were grown to cell density of 2. Techniques vary widely and include lipid-based transfection and physical methods such as electroporation. 5 μg/mL with PEI 7. Expi293F cells transfected with pcDNA-IGG-PGLYRP plasmids were cultivated in 100-ml flasks with a final volume of 30 ml of Expi293 culture medium. Methods: The coding sequence of mature KLK9 isoform (mat-KLK9) was expressed in an Expi293F mammalian system and the synthesized polypeptide was purified through a two-step protocol. All contents of Gxpress 293 Transfection & Optimization of cell culture medium, cell line, and transfection enhancers: (A) Expi293F Cells were seeded at 0. We transfected 1 μg plasmid DNA per ml of cells according to the instructions of the ExpiFectamine 293 Transfection kit (Thermo Fisher). Viral neutralization was measured by HAI assay. VCD 6 x 10 6/mL 10 x 10 6/mL Cell density for transfection 3 x 10 6/mL 6 x 10 6/mL Time to Protein 5-7 days 8-14 days Additional Cell Lines Expi293F (cGMP) Expi293F cells: Transfections were performed using the ExpiFectamine 293 Transfection Kit (Gibco) following the manufacturer's protocol. The TransIT-PRO® Transfection reagent and kit use animal origin free components designed for high and reproducible protein yield in suspension CHO and 293 derived cells. Learn how to produce recombinant proteins with the Gibco™ Expi293™ Expression System and achieve gram per liter protein yields in a transient HEK 293 system . At 120 h after transfection, the cell culture was centrifugedat130 gfor10min. The Expi293™ Expression System facilitates large-scale transfection of suspension 293 human embryonic kidney cells in a defined, serum-free medium for expression of proteins and virus. Cells were cultured according to manufacturer’s TransIT-PRO Transfection Reagent and PRO Boost Reagent are comprised of animal-origin free components that are compatible with many chemically defined media formulations. Healthy Doubling Times: • Sf9 cells double every 72 hours • Sf21 cells double every 24 hours. - Cooperated in bacterial BL21 (DE3) Tau-protein productions; Optimized production methods (scale-up, medium screening, induction schedules, harvest and quantification methodology, SDS-Page). IgG 3-Fc production was assayed at different 2 vials frozen Expi293F cells 1 bottle 1000 mL Expi293 Expression Medium 1 ExpiFectamine 293 transfection kit for 1 L of culture 1 vial Antibody Expressing Positive Control Vector 1 bottle 100 mL Opti-MEM Reduced Serum Medium Store all components at +2°C to +8°C, except frozen cells are stored in liquid nitrogen: Expression System: Mammalian It is designed for transient transfection of high density cultures of HEK (human embryonic kidney) 293 cells. Component Amount Expi293F™ Cells 2. • Implemented polymer based transfection protocols for siRNA and evaluated RNA interference-mediated gene silencing (VEGF and HIF1A). 8 μL ExpiFectamine 293 reagent was added to 140 μL Optiplex and incubated for 5 min before addition to 150 μL Optiplex containing 2. 5 ml of cells per condition was treated with the respective ligands (10 μM) at 37°C, and 2 ml of pelleted cells per The coding sequence of mature KLK9 isoform (mat-KLK9) was expressed in an Expi293F mammalian system and the synthesized polypeptide was purified through a two-step protocol. Thus, we have developed a protocol for the generation of pure PGLYRPs in Expi293F cells. 9 × 10 6 cells/ml in 8. At 4–6 days post-transfection, supernatants were cleared by low-speed centrifugation (900 rpm, 4°C, 5 min) and incubated with Ni-nitrilotriacetic acid (NTA) resin (Invitrogen) according to the Transient transfection typically requires 10–15 days from culture expansion to purified protein (Figure 2). All three cell lines were transfected, at the 20-mL scale, to produce CL40-MND-GFP. (a) Viable cell density and (b) cell viability posttransfection (>70%). Sf9 cells (Invitrogen) were transfected with Bacmid DNA using jetPRIME® transfection reagent (PolyPlus transfection, catalog no. integrates seamlessly with high throughput, automated transient transfection through to high throughput, automated protein purification. With plasma, there are a lot of fibrins and fibrinogens that also get precipitated with ExoQuick. HEK293 Protocols: Cell Culture, Transfection, Protein Production HEK 293 cells are popular for their ease of growth and transfection ( HEK293 Transfection Kit ), making them a common cell culture in cancer research. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost). Cell concentration and viability ere monitored every day and the cultures were harvested at 3 days postw - transfection. (NYSE: TMO) is the world leader in serving science, with revenues of $17 billion and approximately 50,000 employees in 50 countries. A typical, finalized manual protocol In an effort to share best practices for maximizing protein expression in transient and stable systems, we talked to Rich Altman and Matt McKenna, Field Application Scientists supporting the Expi Protein Expression Systems at Thermo Fisher Scientific, about maximizing protein expression and achieving high titer in Expi expression systems. After transfection, allow cells to grow and to express the protein for G418 resistance under non-selective conditions for about 24-48 hours (reach the peak of protein expression according to your transfection system). For Research Use Only. protocol for this technique can be found in Adherent Cell Culture, page 13. 0 × 106 cells/mL Incubate for 24 hours at 37°C Expi293F™ Cells 7. HiTrap rProtein A column (GE Healthcare) was used to purify antibodies from Plasmid DNA was transfected into Expi293F cells (ThermoFisher, A14527) using the Expi293 Expression System (Life Technologies, A14524) according to the recommended proto- col. Not for use in diagnostic procedures. 3. 89129-924), and recombinant baculovirus particles were recovered. e. 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 Expi293F™cells were seeded following the recommended protocol in Expi293™ Expression Medium, and transfected with FectoPRO® + FectoPRO® Booster (0. expi293f transfection protocol