Electroporation makes the membrane more permeable transiently, allowing DNA to enter the cell. Place SOC recovery medium in a 37°C water bath. Qian Chen and Massimo Gadina. The Jurkat cell line was established from the peripheral blood of a 14 year old boy by Schneider et al. 5 μM anhydrotetracycline (aTc; Cayman Chemical) and were selected beginning 24 h post-transfection with Blasticidin Figure 1 CAR-T cell transfection approaches (Creative Biolabs) Electroporation is emerged as a powerful tool for the genetic modification of diverse cell types based on the transient disruption of cell membrane via exposure to an electric field, which allows charged molecules to enter the cell. The line was cloned from cells obtained from Dr. Our results suggest that electroporation of DNA is more toxic than mRNA transfection. mirusbio. VWR #60818-667) at room temperature. Many robust EGFP signals suggest high transfection efficiency. Jurkat E6-1 cells were washed 2 times in OptiMem (Thermo Fisher, Waltham, MA) (without antibiotics) before being resuspended in OptiMem at 1x10^6 cells/ 100 μl. 4. 5% vs 7. g. 5 µM of Alt-R Cas12a RNP, together with 3 µM of Alt-R Cas12a Electroporation Enhancer and 3 µM of single-stranded DNA template (Ultramer oligos, IDT) were electroporated into Jurkat cells using the Neon Transfection System (Thermo Fisher). IMPORTANT! Avoid creating bubbles, which can hinder electroporation. 0 µg of siRNA Earlier attempts to apply CRISPR/Cas9 for gene editing in primary human T cells used either viral delivery of Cas9 and gRNA (Wang et al. e. Optimization of conditions for each cell line is essential. ), cationic lipid-mediated (lipofection) and/or physical (electroporation, cell squeezing, direct microinjection, magnetofection etc. 7, THP-1 cells. An experimental study has been performed for cell suspensions to compare efficiency of cell transfection. The resuspended cells were transferred to cuvettes and immediately electroporated using the program X-005. Inspired by the validated and recognized magnetic drug targeting technology, this original method is a revolution for transfection and infection. Nuclease 3NLS complexed with Alt-R CRISPR-Cas9 crRNA and tracrRNA) in the presence of Alt-R Cas9 Electroporation Enhancer, on the Amaxa 4D and Bio-Rad Gene Pulser platforms. 4308 915. Split cells, if necessary, to obtain optimal confluency for electroporation. 125–4 μM RNP (Alt-R S. Finally, preliminary experiments were carried out with primary T cells from two different donors. The device was used to reversibly electroporate a GFP plasmid (pMaxGFP) in CHO - K1, C2C12 and Jurkat cel ls, with transfection efficiencies better than or similar to previous flow - through designs. Many scientists are shifting toward the use of cell types that are more relevant to in vivo applications, including primary cells, which are considered difficult to transfect. The DNA is the only thing about the electroporation itself that customers need to optimize, Brady says: “High DNA will give high transfection efficiency, but it will lower your viability, because putting in a lot of DNA is toxic to the cells. 53±0. initiate the transfection [22]. Schematic representation of common transfection methods. , Jurkat cells) Use of Alt-R ® genome editing reagents and electroporation are key Use the conditions presented here for Clone E6-1 Jurkat cells as a starting point for optimization of CRISPR reagent delivery in cell types requiring electroporation. 2 × 10 7 cells in cytomix media were co-transfected by electroporation at 175 V, 72 ohms, and 1800 microfarads. Kendall Smith and are mycoplasma free. Introduction: GenMute™ Reagent is a novel biodegradable polymer based siRNA and DNA transfection reagent. Jurkat T cells were transfected with 0. Transfection medium was replaced with fresh cell culture medium 20 h post-transfection. The most common systems currently used for relatively efficient molecular delivery to these cells are expensive electroporation instruments. It is a powerful tool for transfecting large DNA fragments and achieving good transfection efficiencies in cell lines. After electroporation cells were plated in media that contained either 30 µM of Alt-R HDR Enhancer (gray bar), or equal volume of DMSO The toolbox has three different Jurkat cell lines expressing distinct Cas9 variants, including wild-type Cas9, dCas9-KRAB, and sunCas9. 3 HIV-1-based vector is shown. For each transfection, 100 μg of donor vector and 50 μg of pAIO were transfected into early ring-stage parasites in 2 mm gap electroporation cuvettes (Fisher) using a BioRad Gene Pulser II. View specifications, prices, citations, reviews, and more. 51% vs 53. 4ml £325. Electroporation transfection method was developed 35 years ago 13,14. In the past many improvements have been made, for example, the nucleofector technique 15 , 16 that provided the optimization we show that nanochannel electroporation can deliver precise amounts of a variety of transfection agents into living cells. Incubation intervals on ice are beneficial Magnetofection™ is a simple and highly efficient transfection method to transfect primary cells and hard to transfect cells. p. . 2013;7:4351-8 112. io also provides an interactive version of this protocol where you can discover and share optimizations with the research community. cell line (Jurkat cells) at viabilities close to 90%. We demonstrated that the toolbox allows us to rapidly disrupt endogenous gene expression at the DNA level and to efficiently repress or activate gene expression at the transcriptional level. 5 1 Transfection efficiency with GFP plasmid after 24 hours is over 80% Human PBMC Transfection of CAR-T2A-GFP with SB Transposon CAR GFP 60. Jurkat E6-1 (A), K562 (B), Raji (C), or Raw 264. Department of Microbiology and Immunology, Queen’s University, Belfast, Northern Ireland. The device consists of two microchannels connected by a nanochannel. a A representative flow cytometry plot of Jurkat cells transfected with pmCherry-C1 plasmid DNA and subsequently virally transduced with ZsGreen pnl4. 00% 02-3 1648% 00 102 02-2 0. Suspended (Jurkat) and adherent cells (10T1/2 and Huh-7) were tested. An HCD transfection of Jurkat cells can thus be used to efficiently transfect at least up to 2 × 10 9 Jurkat cells at a time with tolerable mortality and should easily be scalable beyond this point by keeping the polymer density at 3 µg per 10 6 cells. et al. Transfection e ciency % 100 90 80 Gene Pulser electroporation buffer is recommended for mammalian cells especially difficult to transfect cells including primary and stem cells. 7 (D)cells were electroporated with an EGFP reporter plasmid at various voltages using the Ingenio® Electroporation Solution (Mirus Bio) and the Ingenio® EZporator® Electroporation System (Mirus Bio) using either 0. 8±1. atio of transfection reagent to nucleic acid R Here we report efficient, large volume transfection results by using a scalable-volume electroporation system. Multiporator Transfection Protocol Protocol No. Sep 30, 2019 · NEI provides twofold higher net transfection efficiency than biochemicals and electroporation in Jurkat cells. To represent all possible electroporation methods used in the transfection, we used DC and AC electric fields. Different cell confluencies, DNA/reagent Jurkat E6-1 (A), K562 (B), Raji (C), or Raw 264. Transfectants were maintained in 0. Genome editing efficiencies were determined by target amplification followed by next-generation sequencing on an Illumina instrument. Apr 14, 2009 · Alternative transfection approaches, including electroporation and virus-mediated siRNA delivery (6, 7), have been used; however, these methods can be cytotoxic or perturb cellular function in unpredictable ways. Electroporation is a physical transfection method that permeabilizes the cell membrane by applying an electrical pulse and moves molecules via the electrical field into the cell. Cell Line Specific Transfection Electroporation Gene Expression In Vivo Delivery TransIT®-Jurkat Transfection Reagent Order #: MIR2126. Cas9. Jurkat T cells (107 ) were transfected with 25 μg of pcDNACIITA-6 and 1 μg of neomycin DNA by electroporation using Gene Pulser Transfection Apparatus (BioRad, Richmond, CA) according to the manufacturer's protocol. Additionally, significantly increased activity for all of the AP-1 transactivation domains except that of junB were demonstrated in Jurkat cells after PMA and ionomycin stimulation. This can be a problem since although dead cells do not form synapses, when they are in excess over the living, transfected cells, they may interfere with the formation of conjugates made by living Th cells. Biolistic Particle Delivery Systems Helios ® gene gun and PDS-1000/He™ systems use a helium pulse to accelerate gold or tungsten particles coated with DNA directly into target cells, including cultures, tissues, plants, and more. 4%) for Jurkat lymphocytes (nonprimary cells), and sonoporation was better in terms of viability (64. Cell lines were prepared at a density of 2 x 105cells per 10 µL tip, for electroporation in Buffer R (component of Neon Transfection System Kits) with 1–1. Other buffers may be sufficient as well. Stocks of viruses were produced by chemical transfection of the human kidney fibroblastic cell line 293T, or by electroporation of Jurkat-T cells. Alt-R CRISPR-Cas9 system—RNP electroporation, Neon Transfection system (862 KB) Alt-R CRISPR-Cas9 system—RNP electroporation, Neon Transfection system (862 KB) Alt-R CRISPR-Cas9 system—RNP electroporation, Gene Pulser Xcell system (503 KB) Ingenio® Electroporation Solution - The High Efficiency, Broad Spectrum Solution For Nucleic Acid Delivery into Primary Cells and Hard to Transfect Cell-lines A. Analysis of cell doubling time, intracellular calcium levels, and messenger ribonucleic acids (mRNA) expression changes after these gene delivery methods reveals that viruses and electroporation adversely affected cell behavior. 45 μm, Sarstedt), and immediately used. ). 020 11/1999 Cell line Jurkat, T-lymphocyte, human leukemia (suspension cell line) Transfection with Plasmid pEGFP-N1 (in bidistilled H 2 O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI 1640 / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Prof. 2 cm or 0. No. The Cell Line NucleofectorTM Kit V is for transfection of cell lines, e. , 2013). Prior to electroporation, 1 ×10 6 Jurkat cells were centrifuged at 90 g for 10 minutes, resuspended in 100 µL of the desired electroporation buffer and mixed with 4 µg of pT2-GFP plasmid. Total Jurkat cells (ATCC® TIB-152™) were transfected with program CL-120 and 1 μg of pmaxGFP™ Vector in 20 µl Nucleovette™ Strips. Alternatively, protocols are provided for reverse transfection in 96-well plates and 384-well plates (see the HiPerFect Transfection Reagent Handbook ). Please select at least one, or any combination of filters below to refine your selection and click Search. D): High magnification image of Figure C (x40). The cells were then electroporated using the Lonza 4D-Nuncleofector X Unit with the SE Cell Line 4D-Nucleofector Kit1 accord- Transfection of Jurkat cells with METAFECTENE. Jurkat cells were maintained in RPMI 1640 media supplemented with 10% fetal calf serum and antibiotics at 37 °C, 5% CO 2. Lauer and B. Successful CRISPR genome editing in hard-to-transfect cells (i. 3% 14 days CAR-T2A-GFP after 14 days 02-1 0. Chemical methods neutralize the negative charge of DNA, facilitating its uptake. VCA-1003 Cell Line Nucleofector™ Kit V For efficient transfection of cell lines e. Use an appropriate electroporation system to transfect electroporate the Jurkat cells; we have had success in using the Neon™ system. Unit: 10 x 1 ml. Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. - Electroporation Cuvettes - Cuvette Chamber & Stand Holder - Laser Thermal Microinjector - Mechanical Vibration Units - Ultrasounic BioMicroscope - KTAC-4000: Sonoporator - LF101: Cell Fusion Device - LF201: Cell Fusion Device - Electro Cell Fusion Electrodes - Electroporation and Electro Cell Fusion Accessories - Electroporators Transfection of suspension cells, such as Jurkat-FHCRC Cells, has always been challenging, both technically and financially. Gene Pulser electroporation buffer can yield both high transfection efficiency and high viability in difficult cell lines. Resuspend in 20 µl serum-free RPMI by incubation @ 65C. 5 and 1 is shown (n = 5). Transfection of both HEK293 and Jurkat cells with dCas9-activators showed that regulatory regions downstream and upstream of FOXP3 promoter can be very Flow Electroporation® technology is based on a fundamental principle of cell membranes ­– the reversible permeability of membranes in the presence of an electrical field – thereby creating a universal transfection technology capable of high-performance delivery of virtually any molecule (s), including DNA, RNA, proteins and cell lysates, to any cell type with minimal cell disturbance. This constitutes a particular promising approach for efficient transient transfection at large scale. After electroporation, electroporated cells were stained with fluorophore molecules that are representative of apoptotic and necrotic-based cell death. 1. Optimize Transfection Performance For All Reagents, Cells and Nucleic Acids 1. It was demonstrated that electroporation was superior to sonoporation in terms of viability (65. Change the cell culture media on the cells 1 day before electroporation. 3% vs 50. 4 µM of Alt-R Cas9 RNP targeting multiple sites in the human genome were delivered into human Jurkat cells via electroporation using the Neon Transfection System (Thermo Fisher), along with 4 µM of Alt-R Cas9 Electroporation Enhancer and 3 µM of single-stranded DNA template (Ultramer oligo, IDT). com Primary cells as well as certain cell-lines are known to be refractory to traditional chemical transfection methods. For adherent A549, U2OS and N2A cells, 5 × 10 4 cells were used per electroporation. I'd highly recommend trying Xfect, chemical transfection reagent, as it is far better than any of the other chemical transfection reagents we tried. 5ul Optimem (per transfection point) - In another tube mix 1ug of plasmid and up to 25ul with Optimem - Add the lipofectamine mixture to the DNA tube, slowly and pippeting up and down. For lipid mediated transfection or electroporation by Neon instrument, we tested Lipofectamine 3000, RNAiMAX or MessengerMAX in each listed cell line with each cas9 version (plasmid, mRNA or protein). The MaxCyte STX uses proprietary flow electroporation technology with a library of pre-loaded protocols to transfect CHO, HEK, CAP-T, Jurkat, Vero, insect cells, primary cells, stem cells, difficult-to transfect cells, and other cells commonly used for protein production and cell-based assays with a variety of biomolecules including DNA, RNA, siRNA, proteins, or other molecules of interest. Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. 00% 8362% CD8+ CARGFP 65. Ulrich Zimmermann Lehrstuhl fr Biotechnologie Biozentrum Universitt Wrzburg dilute your cells the day before transfection also Raji cells must be perfectly fit and healthy, otherwise you waste your time • suspend 15 µg plasmid DNA with 200 µl pure RPMI in 4 mm electroporation cuvette • 10 Mio Jurkat cells per sample • spin, resuspend in 300 µl pure RPMI Compare Jurkat Cell Line Transfection from leading suppliers on Biocompare. 4 h after transfection, 1 μg/ml PHA-P and 50 ng/ml phorbol 12-myristate 13-acetate were added to enhance . 200,000 cells were sus-pended in 20 ul of transfection reagent and 2 μgof pmCherry-C1 plasmid DNA was added. Electroporation with pEGFP-N1 using the same pulse conditions resulted in GFP-overexpression in 62% of Jurkat or 9% of K562, with survival rates of 50% and 40 %, respectively. But as with other transfection methods, the optimal conditions When compared to ectopic expression of FOXP3 via plasmid electroporation, upregulation of endogenous FOXP3 via the Cas9-based method resulted in prolonged expression of FOXP3 in Jurkat cells. 9% 14 days Electroporation----- Select by cell line: Lipid Protocols Electroporation-based transfection. Harvest Jurkat Δ76 cells, wash with PBS and count. In this study, the Gene Pulser MXcell system was used for screening and determining optimal electroporation conditions for Jurkat cells. Boukany PE, Morss A, Liao W. Neurites are shown clearly. , GFP-CD63) in the Jurkat clones (Video 1), a considerable fraction of cells may die after electroporation. Jun 28, 2016 · A human T lymphoid cell line, Jurkat, was infected with a lentivirus vector followed by transfection with the TALEN–HIV by electroporation. - Incubate 30 min at RT TransIT®-Jurkat Transfection Reagent – MIR 2124 0. Transfection of B- and T-cell lines by electroporation The following are general guidelines for transfection of non-adherent B and T cell lines that I have done in the past. Cell viability was determined as % PI negative cells and is usually around 80% after 24 hours. KEYWORDS:Electroporation, Gene delivery, Microfluidic RNP complexes (4 μM) were delivered into Jurkat and HEK-293 cells via a Nucleofector™ system (Lonza) in the presence of Alt-R Cas12a (Cpf1) Electroporation Enhancer. Find transfection solutions for efficient delivery of DNA, siRNA, oligos, and RNA into adherent and suspension cells, including hard-to-transfect cells. We offer a range of Xfect transfection reagents that are known for superb performance, low toxicity, and user-friendly protocols. - in a luminometer tube mix 2. Widely used in gene therapy, cancer treatment, fusing cells together, transferring plasmids directly from one cell to another, and producing gene knockout systems (RNAi), electroporation is an incredibly valuable technique. Jurkat, Clone E6-1 cells (ATCC® TIB-152™) were transfected with the Cell Line Nucleofector® Kit V, Program X-001 and 2 µg of Select by cell line: Lipid Protocols Jun 12, 2017 · In this experiment, T cells (Jurkat cell line) are cultured to reach a suitable density for transfection and then re-suspended in electroporation buffer. Broad application of CRIPSR/Cas9 for gene editing in primary T cells has been hampered by limitations in transfection efficiency and the requirement for TCR stimulation. High transfection efficiency of Jurkat cells with Neon Transfection System. 4 cm electroporation cuvettes. 1. ACS Nano. Electroporation: An electrical pulse creates pores in Jurkat Acute T-cell leukemia, human ++ A transfection reagent with low toxicity is ideal for all gene Transfection is most commonly achieved by one of three methods: chemical-based (calcium phosphate, polymers (DEAE-dextran, cationic), activated dendrimer, etc. The Gene Pulser MXcell electroporation system can be used to quickly optimize electroporation conditions, which can then be used in the overall scientific design. 7 (D) cells were electroporated with an EGFP reporter plasmid at various voltages using the Ingenio® Electroporation Solution (Mirus Bio) and the Ingenio® EZporator® Electroporation System (Mirus Bio) using either 0. 3s pulse interval at 0. 3. Alt-R HDR Enhancer improves HDR mediated by S. Electroporation For suspension cells, such as Jurkat T cells, K562 cells or CD34+ human cord blood cells, 1–2×105cells were used per electropo- ration using Neon® Transfection System 10 L Kit (Thermo Fisher Scientific). Prepare 17 mm x 100 mm round-bottom culture tubes (e. Transfection with HiPerFect Transfection Reagent can be carried out using the common transfection procedure of seeding the cells 24 hours before transfection. s. With our proprietary pH Dependent Conformational Change (PDCC) technology, the biodegradable polymer was chemically modified by addition of pre-screened hydrophobic groups to side chain, making GenMute™ Reagent the most powerful siRNA delivery tool. For electroporation, 800 μl flagellate suspension was mixed with 10 μl plasmid DNA in an electroporation cuvette and incubated at room temperature for 3–5 min. Lipid-based reagents can also coat DNA while forming micelles. , 2015) or transfection by electroporation of gRNA/Cas9 expression constructs (Mandal et al. physical or direct transfer methods such as electroporation or chemical mediated transfection. This comprehensive guide covers all of the different transfection methods and discusses the best method to use in every sample type. 8±2. 00 Unique Formulation - Maximize transfection performance in Jurkat cells and other hard-to-transfect cells such as K562, RAW 264. Over 90% transfection efficiency was achieved for human PBLs and murine T cells stimulated with ConA or antigen-specific pepetide [1]. , 2016). We have spent more than a decade to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs), and subsequently for T cells, B cells, bulk PBMCs, and several cell Transfection with HiPerFect Transfection Reagent can be carried out using the common transfection procedure of seeding the cells 24 hours before transfection. Electroporation works well with cell lines that are refractive to other techniques, such as calcium phosphate–DNA coprecipitation. Electroporation is extremely useful for difficult-to-transfect cell lines and primary cell transfection. Dec 01, 2007 · The final gene silencing experiment involved the electroporation of Jurkat cells with two specific functional Cdc2 siRNA molecules using optimized electroporation parameters and 1. Intracellular uptake of reporter vector encoded with EGFP at 24 hr following transfection of Jurkat cells with Neon Transfection System. 15338-100). 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control. Jurkat cells were transfected by electroporation in cuvettes. Gopalakrishnan Mirus Bio LLC – www. For lipids, we listed the reagent that resulted in best cleavage efficiency reported in Table 1. 1mL/s of flow rate) in the presence of DNAplasmid encoding eGFPreporter gene (80 µg/mL) driven by a CMVpromoter. Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. Transfection of eukaryotic cells used is widelyto study gene functions and gene regulation. I also will try to Celetrix was founded in 2012 in Virginia to commercialize new tyes of high effiiciency electroporators . Then click on the protocol link in the results to view the associated PDF file. These findings suggest that RNA electroporation is preferable for experiments aimed at investigating the role of HTLV-1 gene products in the context of primary T-cells, which Nanostraw-Electroporation System for Highly Efficient Intracellular Delivery and Transfection. Protocol. Oct 18, 2018 · Gene editing in primary T cells not only represents a valuable research tool, but is also critical for next generation immunotherapies, such as CAR T cells. Figure 1. 5, 5, 10, 15, 50 mL, respectively, were flow elec- trotransfected with 4 pulses/cell at 1 kV/cm, 400 µs pulse width (2. Genomic DNA was isolated 72 hr after transfection. Electroporate using the Neon ™ Transfection System . Jurkat-T cells were electroporated in 2-mm gap cuvettes with a BTX electroporation system set to deliver 20-ms pulses at 150 volts. Cas12a (Cpf1) Nuclease V3. physical technique, electroporation may, in theory, be appli-cable to any cell types, its efficiency depending only on the size of the cell and dielectric properties of cell components. You need to pre-optimize (1) the electroporation parameters and (2) the selection paratmers. . 5 mL prewarmed medium in a 24-well plate. The fusion constructs and the reporter plasmid are now being transiently transfected into resting murine A. Transfection System is a novel, benchtop electroporation device that employs an electroporation technology by using the pipette tip as an electroporation chamber to efficiently transfect mammalian cells including primary and immortalized hematopoietic cells, stem cells, and primary cells. General transfection techniquesclude introduction in genes of into cells with calcium phosphate co-precipitates, or by electroporation, in which a brief electric pulse transiently creates holesin the cell membrane. Jurkat-FHCRC Cell Avalanche™ Transfection Reagent (human leukemia cell) Transfection of suspension cells, such as Jurkat-FHCRC Cells, has always been challenging, both technically and financially. HepG2, HL-60, Jurkat, K-562 , MCF7, SH-SY5Y, or THP-1. The Ingenio EZporator ® Electroporation System offers: Alt-R HDR Enhancer improves HDR mediated by A. The target sequence was destructed in approximately 10–95% of the p17 polymerase chain reaction clones, and the efficiencies depended on the Jurkat–HIV clones. Mar 30, 2017 · Results showed that the RNA transfection technique combines a high transfection efficiency with low toxicity, not only in Jurkat T-cells but also in primary T-cells. - Bacteria / Yeast Electroporation - SP100: Sonoporator - NEPA21: Illustrated Applications - NEPA21: Publications by Research Application - Connection Cables - CU700: Monitoring System - CU902: Polarity Exchanger - CUY21 EDIT Electroporator - CUY21 EDIT-S Electroporator - CUY21 SC Electroporator - In Vivo, In vitro Electrodes - Electroporation When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. The 24-well optimization protocols were performed using the 10 µL Neon tip, and the cells were dispensed into 0. 2 steps pulse electroporation using the electrodes (CUY900-13-3-5) for adherent cells: B): EGFP fluorescence image of the neurons 2 days after electroporation: C): High magnification image of Figure B. The agent also promotes pDNA uptake when simply added to a mixture of cells and pDNA. 2016;8:243-52 113. After electroporation, cells were plated in media that contained either 30 µM of Alt-R HDR enhancer (gray bar), or equal volume of DMSO solution as the negative control (light 4. Chang L, Bertani P, Gallego-Perez D. The Ingenio ® EZporator ® Electroporation System is ideal for mammalian and insect cell transfection and is optimized for use with our Ingenio ® Electroporation Solution. Cite Electroporation Instruments Find, compare and review different cell electroporation instrumentsSearch. Pipette 10 μL of the T-cells mixed with Cas9/gRNA complexes into the Neon ™ -μL tip. For adherent A549, U2OS and N2A cells, 5×104cells were used per electroporation. 5ul lipofectamine and 22. Jurkat electroporation. 5 µg of DNA encoding GFP. High-performance, cost-effective electroporation for hard-to-transfect cells. 2 Co-electroporation of Jurkat Δ76 with TCR and NFAT reporter (Day 2): Preheat 4 mL RPMI + 10% FBS with no antibiotics medium per well in 6-well plates. Nanoscale. Transfection System. 4%) with the advantages of superior cell viability, comparable throughput, and user-friendly interface. The cell to be transfected is positioned in one microchannel using optical tweezers, and the transfection agent is located in the second microchannel. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. 5 Add DNA solution to cells in cuvette and mix. , and was originally designated JM. HepG2 HL-60 Jurkat K-562 MCF7 SH-SY5Y or THP-1 in the Nucleofector™ 2b Device SBA-1006 Nucleofection Wet Lab Training Training experiment with the 4D-Nucleofector™ System or the 96-well Shuttle™ Add-On Electroporation is a physical method of nucleic acid transfer wherein the cells and nucleic acids are subjected to high voltage electric pulses. High Efficiency Delivery - Achieve 5-10% transfection efficiency in Jurkat cells to ensure experimental success. 2. A high efficiency DNA transfection reagent optimized for Jurkat and other hard-to-transfect cell types TransIT®-Jurkat Transfection Reagent is specifically optimized to provide exceptional  transfection efficiency of plasmid DNA in Jurkat cells and cell types of associated lineage. ” For suspension cells, such as Jurkat T cells, K562 cells or CD34+ human cord blood cells, 1–2 × 10 5 cells were used per electroporation using Neon® Transfection System 10 μL Kit (Thermo Fisher Scientific). Jan 23, 2017 · In a previous work, our group developed “in house” electroporation buffers (termed “Chicabuffers”) that had comparable efficiency with Lonza’s buffers for the transfection of the human T cell line Jurkat and primary T lymphocytes from mouse and human origin (Chicaybam et al. Electroporation was performed by a double pulse program consisting of one pulse at 800 V, 25 μF followed without delay by one pulse at 100 V, 1500 μF. Neon and Lonza electroporation also work. Use cells with the lowest passage number possible. Lipofectamine® LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. For instance, the square-wave pulse-based new Electroporation Protocol (C2986) Protocols. Virus-containing supernatants were harvested 48 h post-transfection, centrifuged (3500 rpm for 3 min), filtered (0. Briefly, cells were transferred into an ice-cold Gene Pulser cuvette (0·4 cm) and subjected to a single pulse at 960 μF and Jurkat cells at the volume of 1. The company has a team of accomplished biomedical scientists that also understand the properties of complex cell-to-cell electrical interactions. This process induces temporary pores in the cell membrane, enabling entry of nucleic acids into the cell. Transfection of Jurkat and 293 T cells does not show significant impacts on susceptibility to viral transduction. This reference provides a recommended procedure to transfect plasmid DNA into human Jurkat Human T-Cell Leukemia Cells (ATCC Cat. 15%) and transfection efficiency (15. These approaches resulted in low targeting efficiencies, and DNA Human PBMC Transfection with GFP Plasmid 0. This approach may hence be convenient in the context of T cells therapy, where large number Jurkat E6-1 B. The RNA electroporation system improved transfection efficiency and reduced or eliminated transfection related toxicity. Preparation of non-viable cells for the experiments. Jan 16, 2018 · Electroporation—the use of high‐voltage electric shocks to introduce DNA into cells—can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. m Electroporation Kit for Jurkat Cells (T-Cell Leukemia Cells, TIB-152) • Electroporation Kit include: electroporation buffer + buffer-specific optimized electroporation protocols • Pre-optimized electroporation protocols are provided for delivery of plasmids (DNA), siRNA, mRNA, and proteins A droplet electroporation system has been successfully demonstrated for transfection of Jurkat T cell with much higher transfection efficiency (66%) than that of conventional system (11. Applications with Electroporation Cuvettes The NEPA21 Electroporator makes it possible to achieve high transfection efficiency and viability without resourse to special buffers for difficult-to-transfect cells such as PRIMARY CELLS, STEM CELLS, IMMUNE CELLS, BLOOD CELLS, etc. Note: For Jurkat cells, optimal cell density is between 1 x 105 and 1 x 106 cells/mL Universal electroporation buffer is provided to increase cell transfection efficiency. b The results of several independent experiments with viral transduction performed with MOIs of 0. Do not use freshly thawed cells for electroporation. 83±3. Many cultured hematopoietic cell lines, such as K562, Jurkat, M2 and myelomas can now be transfected with acceptable efficiency (>20%). 7 Culture cells in 5-10 ml RPMI/10% FCS until analysis 16-48 h later. Optimization experiments using different electroporation A key step of any CRISPR workflow is transfecting the gRNA and Cas9 into the target cells, but different transfection protocols have different advantages. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. When certain gene transduction techniques such as electroporation are used to express fluorescent chimeric proteins (i. The key to obtaining a high transfection efficiency is twofold: product choice and good experimental technique. Once the Nucleofector® Supplement is added to the Nucleofector® Solution it is stable for three months at 4°C. C 0 Store at 4 GenMute Jurkat Cell siRNA Transfection Reagent for. , 2014; Su et al. TIB-152) using Lipofectamine® LTX Reagent (Cat. In our lab we work with Jurkat cells and the best way to transfect them is electroporation (if you don´t use lentivirus) and works very well for IPs, luciferase assays, CD3-activation experiments. , 2014; Li et al. K562 and Jurkat cells were electroporated with a Cas9 protein/gRNA, ribonucleoprotein (RNP) complex, composed of 750 nM Cas9 protein (EnGen® Cas9 NLS, NEB) and 1500 nM pre-complexed two-part gRNA (IDT) targeting PPIB using the Ingenio Electroporation Solution and a Gene Pulser Xcell™ Eukaryotic System. 24 hours post Nucleofection™ cells were analyzed on a FACSCalibur™ [Becton Dickinson]. 8±4. C. B is the corresponding fluorescence image of A. Recent efforts to address the challenge of nucleic acid delivery have resulted in a variety of nucleic acid delivery platforms. E7 T cells by electroporation. 7 Oct 17, 2011 · Scientists report on the development of a nanochannel electroportation (NEP) device that can deliver precisely monitored amounts of either large or small transfection agents into individual living Unique Formulation - Maximize transfection performance in Jurkat cells and other hard-to-transfect cells such as K562, RAW 264. 6 Electroporate @ 250 V, 960 µF. The percentage of GFP positive cells (bar) and viability (dot) using propidium iodide was determined by flow cytometry at 48 hours post-electroporation

Source link