Lipofectamine 2000 protocol 293t
- The day before the transfection, seed 5x106 of freshly thawed1 293T cells. 5 mL vial 15 mL vial. Transfection complexes were prepared by mixing the plasmids in a final SeV and VSV-GFP were gifted by Dr. Plate 293T cells in # 10 cm tissue culture dishes and let adhere overnight. All cells were cultured at 5% (v/v) CO2 at 37°C in a CO2 incubator. - Mix 15 µg of the transfer vector, 15 µg of pLP1, 6 µg of pLP2, 3 µg of pVSV-G in 1. Readout System LightSwitch Luciferase Assay Reagent NBP2-32066 293T cells were transfected by calcium chloride methods. Twenty four hours after transfection, cells from each well was lysed in 300 µl of 10 mM Tris The poly(I:C) packed with Lipofectamine 2000 (A 200 ul total at 2. U937, immortalized BMDMs and HeLa cells were transfected with poly(dA:dT), flagellin or plasmids using Lipofectamine 2000 (Invitrogen SeV and VSV-GFP were gifted by Dr. 2. Use 45 ug of plasmid DNA. invitrogen. U937, immortalized BMDMs and HeLa cells were transfected with poly(dA:dT), flagellin or plasmids using Lipofectamine 2000 (Invitrogen Mix 38 =C2=B5g of DNA (for example, 19 =C2=B5g each of p16sheLL and pYS= EAP (seeNotes 2 and 3 in main pseudovirus production protocol) with 2 ml of OptiMEM-I. Lentivirus was collected 48 hours post-transfection and concentrated by prolonged centrifugation *This protocol is for 10 cm dishes. HEK-293T cells were transfected with GFP vector (pEGFP-N3) by PeneFect™ (left panel) and Lipofectamine 2000 (right panel) respectively. Abstract. Higher densities can lead to spontaneous differentiation. (1) HEK293T cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/ml penicillin, and 100 mg/ml streptomycin under the condition of 37°C, 5% CO 2. 293FT cells are a fast-growing derivative of HEK-293T cell line and, besides 293T cells, commonly used for PROTOCOL. The percentages of GFP-positive cells in the new method, the previous PEI transfection method, and lipofectamine 2000 were 93. R. 10% heat inactivated FBS (Gibco). Lentivirus was collected 48 hours post-transfection and concentrated by prolonged centrifugation at Xinxin Liu, Updated August 2014 pRSV 10 ug Mix Measure concentration pMDL 10 ug VSV 10 ug pLenti‐Plasmid or PLKO. The efficiency of transfection was verified using a plasmid encoding green fluorescent protein. Replace with 10 ml of fresh media 2 hours before transfection. To characterize the reporter system, 24-well tissue culture plates were seeded with 1 × 10 6 HEK 293T cells/well. The cells were cotransfected with mixtures of plasmids using 2 μl of Lipofectamine 2000 transfection reagent (Invitrogen, 11668019) following the manufacturer's recommended protocol. 5 X 105 cell/ml Incubate Lipofectamine 2000 is a popular transfection reagent (cationic lipid formulation), whereas the calcium phosphate method is the most inexpensive means to deliver gene to cells and is perhaps the benchmark for evaluating transfection efficiency of chemical-based transfection methods . 3. Cells were seeded into 96-well-plate for ∼24 h before transfection. mRNA Transfection mRNA can be transfected in a 24-well plate by using Lipofectamine® 2000 SeV and VSV-GFP were gifted by Dr. The 1:1,000 dilution of PEI was most tolerated by the HEK-293T cells with a viability of 55. Lipofectamine 2000 reagent and Lipofectamine 3000 reagent were used to. The transfection of HEK293T cells was performed using Lipofectamine 2000 (Invitrogen). Mix 38 =C2=B5g of DNA (for example, 19 =C2=B5g each of p16sheLL and pYS= EAP (seeNotes 2 and 3 in main pseudovirus production protocol) with 2 ml of OptiMEM-I. fected into HEK293T cells using Lipofectamine™ 2000 (all from GeneChem Co. I've confirmed the insert with Sanger sequencing (primer walking). 45-μm filter. , Ltd. Lipofectamine 3000 Reagent Protocol Lipofectamine 3000. sigmaaldrich. Transfecting 293T cells using Lipofectamine 3000 (24well plate) Day 0. 50% efficiency is ৩ মার্চ, ২০১৪ PCR was run using the following cycling protocol: initial activation Transfection of cells with different Lipofectamine 2000 amounts. Polyjet and Lipofectamine 2000 are good reagents for transfection of 293T cells to produce # Anticipated Results This protocol will produce high titer of lentivirus for functional analysis. Volumes are given on a per-well basis. Transfection P19 cells with Lipofectamine 2000 (The same protocol can be used for 293T cells) 1. 5 µg gRNA T2 plasmids using Lipofectamine 2000 (Invitrogen) as per the manufacturer's protocols. Package Contents. The endocytosis efficiency of MSNs in human embryonic kidney 293T cells was greatly increased using Lipofectamine 2000 compared with controls (P < 0. U937, immortalized BMDMs and HeLa cells were transfected with poly(dA:dT), flagellin or plasmids using Lipofectamine 2000 (Invitrogen Comparison of CaPO 4, Lipofectamine® 2000 and Trans IT-Lenti generated lentivirus. 95 and 84. Transfection was performed using the calcium phosphate–DNA coprecipitation method for HEK-293T cells and Lipofectamine 2000 (Invitrogen) for HeLa cells, according to the manufacturer’s protocols. Lipofectamine 2000TM was used for transfection, according to the manufacturer's instructions. 1. After screened with G418 for 4 weeks, Western ChimeraRNA transfection protocol Part I 1. Methods: Recombinant plasmid pcDNA3. 5 μl lipofectamine 2000 were diluted separately in 100 μl Optimem. 5 g plasmid DNA and 9 lipofectamine 3000 transfection protocol. VVC - 3nd generation VSV. To prepare stocks of (VSV/NG/NanoLuc)-SARS-CoV-2 pseudotype particles, 293T cells were plated at 1. 5 mL Opti-MEM; C) Incubate at room temperature for 5min à Combine the DNA mixture with diluted Lipofectamine 2000; D) After 20 min incubation à Add the DNA-Lipofectamine mix to the cells (1 mLà 5 mL, the total medium will be ~6 mL); 4. A. To generate the Flp-In T-REx 293 MUC1-inducible expression system, the Flp-In T-REx 293 parental cells (Invitrogen) were cotransfected with pcDNA5/FRT/TO-MUC1 plasmids and pOG44 plasmids using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol. We compared the transfection efficiencies of the two methods and Lipofectamine 2000 in 293T cells. ২২ আগস্ট, ২০১৯ 1 μg of ER-localized TRICA-mCherry DNA and 1. HIV CMVeGFP Virus was produced in HEK 293FT cells using either CaPO 4, Lipofectamine® 2000 or TransIT-Lenti Transfection Reagent per the manufacturer's protocol. 25 fold, while maintaining the above ratio of four plasmids. Day 1. The virus-containing supernatant was harvested 24 h and 48 h after medium change, cleared by centrifugation at 2000 rpm and 4 °C for 10 min, and filtered through a 0. (17). Then, lentivirus was produced in HEK293-FT packaging cells by co-transfecting the miR-BART9 All plasmid transfections were carried out using Lipofectamine 293T cells were seeded in 6 well plates to achieve 50% confluence at the day of transfection. Trans. The 293T cells in six-well plates were transfected with 1 μg of purified RNA (vector, L-I, or L-II mut) or poly(I):Poly(C) (250 ng) in Opti-MEM (Invitrogen) using Lipofectamine 2000 for 4 h. 293T cells adhesion and transfection - Looking for cell line for GFP + HSP (reply: 3); CV1 transfection using Lipofectamine 2000 - (reply: 3) ৮ মার্চ, ২০১৬ This protocol is adapted from Ran et al. 25 1. com/content/sfs/man To prepare HEK 293T cells for reverse transfection: 1) 24 hours before needed, plate 10 x 106 cells in 10 ml media in a 10 cm dish. Cells were transfected 20–24 h after seeding using Lipofectamine 2000 (Thermo Fisher) by following the manufacturer’s protocol. 75 µl of Lipofectamine LTX™ Reagent into the above diluted Opti-MEM :DNA solution, mix gently and incubate 30 minutes at room temperature to form DNA- Lipofectamine LTX™ Reagent complexes. At 16 24 h, the cells were transfected with the plasmid DNA for len-tiviral production. After 5 min, add the DNA containing solution with the Lipofectamine™ 2000 containing solution and mix gently. Size 0. Detection using indirect fluorescence of the signal corresponding to staining with anti-FLAG mouse antibody (top) and an antibody (ab5517) against CLLD8 (middle) in 293T cells. Endothelial. For serum starvation, HEK 293T cells were cultured in DMEM supplemented with 1% penicillin/streptomycin (serum-free medium). Gfp expression efficiency compared with either transgenic mice or intrinsic cellular processes. Invitrogen has taken the time to optimize this reagent and the recommend protocol that they provide 50 ng of -STAT3) using Lipofectamine 2000 pGL3 (11668500 , Invitrogen, Carlsbad, CA, USA) which was following the manufacturer’s protocol. 7-shTGM2-B, and pLL3. NSCLC patient samples and immunohistochemistry One hundred and six patients with histologically- The plasmids used were supplied from BEI Resources . Bob has only has one exon and is a relatively short transmembrane protein (515AA). Protocol Outline. U937, immortalized BMDMs and HeLa cells were transfected with poly(dA:dT), flagellin or plasmids using Lipofectamine 2000 (Invitrogen 2000 and Lipofectamine 3000 were used to transfect U2OS and HepG2 cells in a 12-well format. Dual luciferase reporter assay kit (Promega) Protocol. After 24 hours, the firefly and renin luciferase activi-ties were measured by the double luciferase method (Promega, Madison, WI, USA). The DNA to be expressed is cloned into the pMT2T (or related) using Lipofectamine 2000 (Invitrogen) following the manufacturer recommendation. They were used directly on NRVMs at 1 μM, combined with Lipofectamine 2000 (1:10 ratio). Pseudotyped HIV-1 particles were generated by cotransfecting pNL4. Even more differences than between the packaging mixes were found when comparing HEK-293T and 293FT as producer cell lines. Pre-warm 50ml of Optimum (stored in cold room at 4°C) ~10min. before transfection to reach a 50–80% confluence at the day of transfection and then transfected with 35–40 μg through the calcium phosphate protocol as previously described . Xinxin Liu, Updated August 2014 pRSV 10 ug Mix Measure concentration pMDL 10 ug VSV 10 ug pLenti‐Plasmid or PLKO. 4 ± 1. 1(-)/UBE2C successfully constructed in previous time was transfected into 293T cells by lipofectamine 2000. Transient transfections of PC12 cells were performed according to the LipofectAMINE Plus or LipofectAMINE 2000 protocols (Invitrogen). 5% fetal bovine serum) Typically passage cells every 2-3 days For immunofluorescence analysis adherent 293T, HeLa HA, or U2OS cells were grown on coverslips in 24-well dishes. For transient overexpression studies, DNA plasmids were transfected using Lipofectamine 2000 reagent (Invitrogen). Organism Type. Subsequently, cytosolic extracts were For Lipofectamine 2000 transfections, 293T cells were plated as described in complete IMDM lacking antibiotics. 4. 5mgof lentiviral construct (pHRST-IRES-TGM2, pLL3. 5), 150 mmol/L NaCl, Transfection Protocols: I use Lipofectamine 2000 (Invitrogen) for transfection of both siRNA and plasmid DNA into these 293 cell lines. 5 ml of OPTI-MEM in one tube. Using Lipofectamine 2000 CD reagent for transfection provides the following advantages: Highest transfection efficiency in many cell types and formats. China). Up to. , Shanghai, China). PROTOCOL. Aspirate the media from the 293T cells and gently add the transfection mix. It allows the highly efficient transfection of a broad range of cell types, including adherent, suspension, and insect cells, as well as primary cultures. • For 293/A658 gene targeting assays: I use 2 µL of Lipofectamine 2000 in 50 µL SeV and VSV-GFP were gifted by Dr. The HEK 293T cells were transfected at confluence (4x105 cells) in six-well plates. Comparison of CaPO 4, Lipofectamine® 2000 and Trans IT-Lenti generated lentivirus. no. Lipofectamine 2000 (Invitro-gen, Carlsbad, CA) was used to transfect cells with plasmid DNA. I get consistent results using this lipid for my transfections. Brieﬂy, 3 to 4 106 293T cells were plated on 100 mm culture dish 24 hours before transfection. Tissues. 6. 97, 34. Cells transfected with the same amount of Emv were used as control and normalized for transfection efficiency. 5 ml of OPTI-MEM. • For 293/A658 gene targeting assays: I use 2 µL of Lipofectamine 2000 in 50 µL 293T cells were transfected by calcium chloride methods. Specifically, I'd like to know what the general ratios you use for packaging, envelope, and transfer plasmids are. ২৫ ফেব, ২০২০ The other protocols used for producing lentiviruses in HEK-293T Lipofectamine 2000 reagent: primary neurons, siRNA, and. G (Addgene) were cotransfected into 293T cells using 27mL of Lipofectamine 2000 (Invitrogen). Cells were washed with PBS once. 45 μm filter. Transfections with polyethylenimine [linear, molecular weight (MW) 25,000] were performed, using the Lipofectamine 2000 transfection protocol, but instead of Lipofectamine 2000, equal The transient transfection was performed using Lipofectamine 2000 (Invitrogen, Cat# 11668019) as described. 3% BSA without serum. GenScript recommends using Lipofectamine 2000 for all transfections. Thank you for providing such a nice product. Fluorescent activated cell sorting (FACS) was done three days after transfection of HEK 293T cells. I normally incubate my cells in media with antibiotics and serum, and a few hours before transfection I aspirate the media and replace with antibiotic free DMEM with serum. 91, and 100 μl Polyjet ( SignaGen) or Lipofectamine 2000 ( Life Technologies) in 15 cm dishes. *This protocol is for 10 cm dishes. Twenty-four hours later the cells were transfected using lipofecta-mine. 100ng of pmCherry-C1 plasmid DNA in 16 ul of Opti-MEM 1 Reduced Serum Medium (Gibco), was mixed with 2 ul of Lipofectamine 2000 at room temperature. ppt. Mix well by inversion or vortexing. 2 Lipofectamine™ 2000 (LF2000) is a reagent that I have found fits exactly what I need. opti-MEM (Invitrogen) 7. Xfect was better than this Lipofectamine method… I was surprised (shocked!) We will switch to Xfect. 5% calf serum, 2. Protocol Transfection Mirus Bio Llc. U937, immortalized BMDMs and HeLa cells were transfected with poly(dA:dT), flagellin or plasmids using Lipofectamine 2000 (Invitrogen protocol. G pseudotyped lentiviral packaging protocol. To transfect cells with siRNA, follow the protocol as described for DNA but transfection efficiency than Lipofectamine® 2000 and FuGENE® HD reagents for Lipofectamine 2000 was also reported in U2OS cells. HEK293T were seeded in white 96-well plates at a density of 1*10(4) in 75 uL antibiotic free medium supplemented with 10% FBS for overnight. Human. 4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) supplemented with protease inhibitors. Transfection P19 cells with Lipofectamine 2000 (The same protocol can be used for 293T cells) 1; Maintain P19 cells undifferentiated in MEM with 10% serum (7. 5 ± 1. In the mean time, change to fresh media in the 293 T cultures now using DMEM (with 1%FBS) to a total volume of 20 ml. Incubate A and B separately for 5 min at RT. Mix A and B together gently (do not vortex) and incubate for 20 min at RT. Incubate the transfection mix for 20 min at room temperature. 5 3. 5 2 100 mm plates 3 13 150 mm plates 6. HEK 293T cells were transiently transfected according to the LipofectAMINE (Invitrogen) manufacturer's instructions. U937, immortalized BMDMs and HeLa cells were transfected with poly(dA:dT), flagellin or plasmids using Lipofectamine 2000 (Invitrogen Invitrogen™ Lipofectamine™ LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. No apparent in vitro or in vivo cytotoxicity was found for Lipofectamine 2000 combining with MSNs. The transfection was performed using Lipofectamine TM 2000 follow- 293T cells were transfected by calcium chloride methods. Plate two plates; one will be harvested for Western blot (Step 3), the other for metabolite analysis (Step 4). Note: Cell growth was assessed ২৯ সেপ্টেম্বর, ২০১৪ or Lipofectamine 2000 \(Life Technologies) in 15 cm dishes. ১০ জুন, ২০১২ Each T175 should be fed with 32 mls of 293T Media. 1-puro-CMV-TurboGFP™ transfer vector and the Lentivirus Packaging Mix Powered by MISSION® Genomics (1:1 ratio, 2 µg/well) with the following reagents: TransIT®-Lenti (3:1, vol:wt), Lipofectamine® 2000 (3:1), Lipofectamine® 3000 (3:1:1), 25 kDa PEI (6:1), or CaPO4 Comparison of CaPO 4, Lipofectamine® 2000 or TransIT®-Lenti Generated Lentivirus. that the best method was using Lipofectamine™ 2000 with Plus Reagent and an overnight incubation with Opti-MEM®. 293T cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf serum, and incubated at 37 °C in the presence of 5% CO 2. 293T Cells. Gently swirl the plates and shake For immunofluorescence analysis adherent 293T, HeLa HA, or U2OS cells were grown on coverslips in 24-well dishes. Lipofectamine 2000 Transfection Reagent (Invitrogen) and Mirus TransIT Transfection Reagent (Mirus Bio LLC) were used for carrying out transfections following the manufacturer’s protocols. Finally, add the DNA–lipid complexes drop wise onto the 293T cultures. Louis, MO, USA). 038% and 37. Therefore: 1. Let sit at RT for 5 minutes. performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions, or polyethylenimine (Polysciences). 293T cells were co-transfected with Psicheck-2/ IRS1 3′-UTR and Psicheck-2/IRS1 3′-UTR mutation reporter plasmids, miR-183-5p or NC and Lipofectamine 2000. Add 51. 5ul Lipofectamine 3000 with 50ul optiMEM per transfection condition. The 3 ml plasmids–Polyjet complex and 1. Maintain P19 cells undifferentiated in MEM with 10% serum (7. 5% GFP positive cells. Lipofectamine 2000 Reagent. Typically passage cells every 2-3 days. These cells are isolated from human embryonic kidneys (HEK). U937, immortalized BMDMs and HeLa cells were transfected with poly(dA:dT), flagellin or plasmids using Lipofectamine 2000 (Invitrogen Detection using indirect fluorescence of the signal corresponding to staining with anti-FLAG mouse antibody (top) and an antibody (ab5517) against CLLD8 (middle) in 293T cells. I am using Lipofectamine 2000 with optimem to transfect HEK cells, but I haven't had much luck so far. Each reaction mix is sufficient for triplicate (96-well), duplicate (24-well), and single well (6-well) transfections, and accounts for pipetting variations. Lipofectamine 2000 (Lipo 2000) was used as a control; c) Relative GFP expression level in GFP-stably expressing 293T cells after the cells were treated by scrambled siRNA (siRNA-NC) transfection with SSBAP or SSBT copolymers at N/P ratios or treated only by the polymer alone with the amount as same as the case of the SeV and VSV-GFP were gifted by Dr. For 24 well plate: 8e4 cells/well in 0. 5 x 10^6 low passage (less than P20) 293T cells per 15cm dish in 15 ml DMEM. HIV CMVeGFP Virus was produced in HEK 293FT cells using either CaPO 4, Lipofectamine® 2000 or TransIT®-Lenti Transfection Reagent per the manufacturer's protocol. The HEK 293T/17 cells (2 × 105 cells/mL) were seeded into six-well plates in DMEM with 10% FBS till they have a 50 70% confluency. CRL-11268) were We chose Lipofectamine™ 2000 for all transfections as it is a common and easy to use transfection reagent. Plasmids were transfected using Lipofectamine 2000 (Invitrogen) in accordance with the manufacture’s protocol. z Lipofectamine 2000 (11668 Learn more at http://www. Dilute Lipofecamine 2000 with OptiMEM 250ul/well; Transfer the transfection mix (500ul) to 293T cells in Seeding media. I follow the manufacture’s recommendations. Adherent 293T/17 cells were To generate the recombinant viruses, eight plasmids were cotransfected into the mixed culture of 293T and MDCK cells (9:1 ratio) by Lipofectamine 2000. lipofectamine 2000 (L2K) and Fugene HD on HepG2 cells. Invitrogen Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids DNA and RNA into eukaryotic cells providing the following. Seed 2. After transfection, the medium was changed to complete medium (10% FBS, 1% penicillin-streptomycin, and DMEM) with DMSO or CHX (20 μg/mL). Lipofectamine 2000 CD reagent is a proprietary animal origin-free formulation for the transfection of nucleic acids into eukaryotic cells. 4μg/ml or 2μg/ml puromycin (Sigma-Aldrich, St. lifetechnologies. Briefly, 293T cells were plated 16 h. U937, immortalized BMDMs and HeLa cells were transfected with poly(dA:dT), flagellin or plasmids using Lipofectamine 2000 (Invitrogen Transfection Protocols: I use Lipofectamine 2000 (Invitrogen) for transfection of both siRNA and plasmid DNA into these 293 cell lines. were transfected with different DNA transfection reagent per manufacturer's protocols. 293T cells transfected with DAP12 only or together with pcDNA3. The transfection of THP-1 cells was performed using Lipofectamine RNAiMAX (Invitrogen) following the manufacturer’s 293T cells were transfected by calcium chloride methods. com/life-science/functional-genomics-and-rnai/shrna/trc-shrna-products/lentiviral- packaging-mix. 5ug of plasmid with 50ul of OptiMEM and 1ul of P3000 Reagent Polyjet and Lipofectamine 2000 are good reagents for transfection of 293T cells to produce lentivirus. 4 3. Effi ciency and OFP expression were analyzed 72 hours posttransfection and (A) U2OS and (B) HepG2 cells showed 4-fold and 80-fold improvement with Lipofectamine ® DNA Transfection Protocol. -293T cells, growing in DMEM + 10% FCS -Sterile DNA . To avoid contamination, the freezing vials should be immerged in 70% alcohol for 2-3 min to thaw 293T cells. I have used LF2000 successfully with the following cell lines: HeLa, 293, CHO, U2OS, and C33A. . 3 mL vial 0. Finally, the firefly lucif- transfection protocol was followed to include pMAX, a GFP expression plasmid, into HEK-293T cells. 5% fetal bovine serum) Typically passage cells every 2-3 days In the first protocol (Ver. The medium 293T cell line was purchased from Invitrogen, and cul-tured in DMEM medium. Gfp positive notation refer to. Seeding 293T cells at 105 cells/well (24 well plate). Workflow of PCR product based HEK293 cell transfection (96-well-plate format with Lipofectamine 2000). Flow cytometry and cell sorting. After screened with G418 for 4 weeks, Western 5. U937, immortalized BMDMs and HeLa cells were transfected with poly(dA:dT), flagellin or plasmids using Lipofectamine 2000 (Invitrogen Our results suggest that Lipofectamine ® 3000 outperforms Lipofectamine ® 2000 and FuGENE ® HD in transfection efficiency and/or protein expression, which is consistent with the claims of the manufacturer who reported it for HEK-293, HeLa, LNCaP, HepG2, and A549 cell lines, the superiority of Lipofectamine ® 3000. 3 Examples of a Negative Control Simplicable. 5 X 105 cell/ml Incubate Adherent 293T/17 cells were transfected in a 6-well plate with pLKO. 6 μg) of siRNA per 1 μg of DNA. Plate cells so they will be 7090% confluent at the time of transfection. with diluted Lipofectamine and incubate for 20 min at room temperature. The cells were transfected with indicated plasmids using Lipofectamine and Plus reagents (Life Technologies) following manufacturer’s protocol. Experimental result: HEK-293T cells were successfully transfected with pGIPZ-eGFP plasmid using X-tremeGENE™ HP ১৫ জুন, ২০০০ They (both) have the unusual property of being highly transfectable by the following Ca3(PO4)2transfection protocol. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. After 24 h, HEK-293T cells were treated with 0-1 μM W2014-S for 24 h, and stimulated with IL-6 (100 ng/mL, PeproTech) for 1 h or EGF (50 ng/mL, PeproTech) for 30 min. Maintain cell density at 80% confluence or lower. ®For each well of cells, add 0. Protocol for rAAV Production A comparison showing transfection efficiency of PeneFect™ reagent vs. Add 1 ul Lipofectamine 2000 into 30 ul DMEM without serum, mixing well and wait for 5 min at room temperature. Note: The content above has been extracted from a research article, so it may not display correctly. Mix and let stand 20 minutes prior to addition to cells. - In another tube, mix 50 µl of Lipofectamine 2000 in 1. In general a 1:1 molar ratio has worked well for Cas9:sgRNA. Then, lentivirus was produced in HEK293-FT packaging cells by co-transfecting the miR-BART9 All plasmid transfections were carried out using Lipofectamine 293T cells were transfected by calcium chloride methods. Cationic lipid-mediated delivery is a fast, simple, and reproducible means for easily introducing DNA, RNA, siRNA, or oligonucleotides into eukaryotic cells. The HCMV BAC DNA was transfected in the cells using the Lipofectamine 2000 reagent (Invitrogen-ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer protocol. 10ul, Lipofectamine 2000. 293T cells were grown to confluence in T150 flasks and transiently transfected with 60 μg of plasmid DNA and 120 μl Lipofectamine 2000 (Invitrogen) per flask. 5. 5 ml 293T cell suspension w ২০ মে, ২০০৯ The objective of this protocol is to transfect 293T cells and produce high titer of LipofectAmine 2000 (Invitrogen 11668-019). The 293T cells and TZM-bl cells (obtained from ABSL-3 of Wuhan University) were cultured in DMEM (Hyclone) with 10% FBS (Gibco) in a 5% CO2 environment at 37 °C. Split cells into plates at appropriate density . IT ®-Lenti Transfection Reagent. 5 mg/ml) was then used to stimulate the RIG-I/NF-kB LUCPorter(TM) HEK 293T cell line as described in Figure 1A. The day before transfection, 500,000 MRC-5 cells per well were seeded in a 6-well plate and were incubated for 24 h up to approximately 60% confluency. Mix 0. 5% fetal bovine serum). CRL-1573) using Lipofectamine LTX Reagent. Invitrogen Lipofectamine LTX Reagent with PLUS Reagent. Polyjet may be better for transfection of 293T cells to produce lentivirus. This reference provides a recommended procedure to transfect plasmid DNA into HEK 293, human embryonic kidney cells (ATCC No. Seed 2x106 293T cells on a 100-cm tissue culture dish and incubate overnight until cells reach ~70% confluence (~1-2 days). For mitoplast images, HEK-293T cells were co-transfected with mt-mCherry and MFN1-GFP plasmids using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s instructions. 75 mL vial 1. 5ml. GFP+ Expression with Lipofectamine Figure 8. After 24 h, the cells were transiently transfected with 1 µg Cas9 plasmid (Addgene, #41815), 0. Transfection Horizon Discovery. Lentivirus was collected 48 hours post-transfection and concentrated by prolonged centrifugation at 2Optimum amount needed is determined from the protocol (see pages 2–3). ™After 30 minute incubation, add 100 µl of the DNA- Lipofectamine LTX Reagent complexes directly to each B) Dilute Lipofectamine 2000 with Opti-MEM: 50 ml of Lipofeactamine in 0. Hi! I've generated a plasmid containing my protein of interest (let's call him Bob) with Gateway Cloning. 7 ml of Opti-MEM. 7-shTGM2-C), 3 mg of psPAX2 (Addgene), and 1. The concentrated solutions were then added to cultured The plasmids used were supplied from BEI Resources . 5 µg gRNA T1, and 0. Mitoplasts were isolated (as described in ‘Whole-mitoplast recording’) 2 days following transfection, and imaged on the confocal microscope as above. At 24 h post-transfection, the medium was replaced by DMEM contain 0. The next day, the transfected cells were infected with the above-described VSV-G complemented rVSVΔG/NG/NanoLuc virus at an MOI of Polyjet and Lipofectamine 2000 are good reagents for transfection of 293T cells to produce # Anticipated Results This protocol will produce high titer of lentivirus for functional analysis. Transfect 293T cells with appropriate plasmids with Lipofectamine 2000 according to the manufacturer’s instructions. In case of 293T cells, coverslips were pre-coated with poly-lysine (Millipore). Mix lentiviral transfer vector and packaging vectors in 600 ul of DMEM in an eppendorf tube. Add 6l of Lipofectamine2000 (from Biostores) and 100µ µl of Optimum to Mix 1. At 72 h post-transfection, the supernatant was collected and amplified in MDCK cells. packaging plasmids into human embryonic kidney 293T cells using Lipofectamine 2000 (Invitrogen). 293T cells were transfected with vectors for overexpression of flagged CLLD8 using Lipofectamine 2000 transfection procedure (Invitrogen). 293T cells were transfected by calcium chloride methods. If using ১৮ ফেব, ২০১৬ However, significant toxicity from Lipofectamine 2000 was also For the Neon 24-well optimization protocol, 24 µg Cas9 protein and 6 µg PROTOCOL - PLASMID PREPARATION shRNA Lentivirus Production using HEK293T cells The following protocol has been optimized for lipofectamine2000 II) vs. 5 mg of pMD2. U937, immortalized BMDMs and HeLa cells were transfected with poly(dA:dT), flagellin or plasmids using Lipofectamine 2000 (Invitrogen Lipofectamine (2000) (µL) 10 60 135 OptiMEM (mL) 0. In both cases, cells are still actively growing when harvested. Aim: To establish a 293T cell line with stable expression of UBE2C and to investigate the effect of UBE2C overexpression on the proliferation of 293T cells. In the present study, we screened a large set of optimization protocol, 24 μg Cas9 protein and 6 μg. Lentiviruses were produced by reverse transfection of suspended 293T cells using 20 ug lentiviral vector, 10 ug pVSV-G, 20 ug pCMV-dR8. Alternatively, 5x106 cells can be cultured for 2 days before transfection. µ 3. After 48h, the cells were infected with the packaged lentiviruses and cultured for 2days before being selection with 0. Then, lentivirus was produced in HEK293-FT packaging cells by co-transfecting the miR-BART9 All plasmid transfections were carried out using Lipofectamine For transfection with 293T cells, calcium phosphate or polyethylenimine was used when cells were of ~50 to 60% confluency. We've used Lipofectamine 2000 with HEK 293FT/293T, N2A, and Hepa1-6 cell lines, following their standard protocol (for 24 well plates, 500ng of DNA total in 50ul of OMEM, with 2ul of Lipo). Lipofectamine 2000 virus production tips. 3‐1. Then, lentivirus was produced in HEK293-FT packaging cells by co-transfecting the miR-BART9 All plasmid transfections were carried out using Lipofectamine Cationic lipid transfection reagents. A sample protocol is listed here for transfection experiments performed in 6-well plates. To test 293T cells for effects of transfection, cells were plated in a 48-well plate at 50,000 cells per well. 293T cell transfection troubles - Lipofecamine 2000 -. ১ সেপ্টেম্বর, ২০১৭ For transfection experiments, 3x105 293T cells passaged less than 20 times 4 μl Lipofectamine 2000, 2 μl Jet Prime and 3 μl Fugene HD. Combine one tube of the first set (OPTI-MEM + Lipofectamine 2000) with 1 tube of the Figure Legend Snippet: The relative growth rates of HEK 293T and Hela cells in the presence of different gene carrier particles. 1, released on July 4th, 2008), we have introduced how to from 293T cells and contain env-IRES-puro. , Nature protocols, Lipofectamine 2000 transfection reagent (Life Technologies, cat. HEK 293T cells were seeded in 12-well plates at a density of 100,000 cells per well. • Simple Protocol - 2000 Lipofectamine 3000 25kDa PEI CaPO. To generate the recombinant viruses, eight plasmids were cotransfected into the mixed culture of 293T and MDCK cells (9:1 ratio) by Lipofectamine 2000. 1‐Plasmid Measure concentration 293 T packaging cells at 1. 001). Plate add per well (mL) Total will be 6-well plate 0. The 293T cells are transformed with large T antigen. 293T cells were transfected using Lipofectamine 2000 (Thermo Scientific); HeLa HA and U2OS cells were transfected using FuGENE HD (Promega). Total RNA and proteins were extracted from 80% confluent cells in culture dishes. Lipofectamine® 2000 Reagent Protocol 2013-2-Lipofectamine® 2000 DNA Transfection Reagent Protocol Transfect cells according to the following chart. Lipofectamine 2000 (Life Technologies, cat. 3LucR-E- (25–30 μg) and the VSV-G envelope expression Briefly, HEK 293T cells were transiently transfected with third generation lentiviral vectors using Lipofectamine 2000. Add 100l of Optimum to the Eppendorf tube to dilute the DNA and mix by µ tapping. 75 29. The numbers of GFP-positive cells were quantified by flow cytometry after transfection. Hi everyone, I'm relatively new to viral transduction, and I'm looking to see if anyone has experience with Lipofectamine 2000 based lentivirus production in 293T cells who can assist me. 1 were used as negative controls. 11668-019) or other preferred transfection reagent Cell line of interest (human embryonic kidney (HEK) 293T cells (ATCC, cat. Procedure: These cells, an Ad5 transfromed human embryonic kidney cell line containing a copy of the SV40 large T antigen, can be transfected very efficiently by the calcium-phosphate method (75-100%!). Transfection of siRNAs and other cell lines was performed with lipofectamine 2000 (Invitrogen) with the manufacturer's protocol. 5 ml fresh DMEM without serum into cells. Transfection of NIH3T3 cells, Hela, Swis 3T3, 293T with Lipofectamine 2000 1. SeV and VSV-GFP were gifted by Dr. Incubate 5 min at RT (Optional) During incubation, change media on cells. Thermo Scientific ™ Nunc 24-Well Cell-Culture Treated Multidishes 142475 Invitrogen ™ Lipofectamine 3000 Transfection Reagent L3000008 Gibco ™ Opti-MEM I Reduced Serum Medium 31985062 Step Tube Complexation components Amount per well (24-well). After 5 min, diluted DNA was ২০ এপ্রিল, ২০১৮ transfection reagents including FuGENE 6, Lipofectamine 2000 and Lipofectamine 3000 in different cell lines and primary cultured cells. Transfection of 293T was performed with a standard calcium phosphate precipitation method . Kidney. In a separate tube mix 85 =C2=B5l of Lipofectamine 2000 with 2ml of Opt= iMEM-I. Place each into 2. 75-1. Add the mix to the plates dropwise, and swirl. Transient 293T cells were transfected by calcium chloride methods. Plasmids should be free of contamination. Primary neonatal mouse cardiomyocytes (NMCMs) were isolated from P1-P3 PHD2/3_HWT and PHD2/3_HKO, following the same kit and instructions that used for NRVMs. Lipofectamine 2000 (Invitrogen, CA, USA) was used to co-transfect the luciferase reporter plasmids and miR-136-5p mimic or mimic negative control into 293T cells. CRL-11268) were 293T cells were transfected by calcium chloride methods. Western blotting The cells were grown to 80~90% confluence and then Transfection P19 cells with Lipofectamine 2000 (The same protocol can be used for 293T cells) 1. lipofectamine 2000 (Invitrogen) 6. 2 × 10 7 293T cells were plated in 15-cm dishes, and transfected the following day with 12. Add ~2g of DNA to Eppendorf tube in the hood. Use 144 ul of Lipofectamine 2000. a leading product, Lipofectamine 2000 on HEK293FT cells. com/transfectionOptimized protocol for Lipofectamine LTX & Plus reagent:http://tools. Stable overexpression and silencing were obtained by transducing MDA- MB-231 cells, HEK 293T, HEK 293, RCC4, RCC4/VHL, 786-O, 786-O/VHL and H1299 cells with retroviral or lentiviral vectors. Briefly, HEK 293T cells were transiently transfected with third generation lentiviral vectors using Lipofectamine 2000. 16 Plasmids for Myc- Ajuba, Flag-Twist and/or their truncation mutants were transiently transfected into 293T cells. I am transfecting in a T75 flask. Invitrogen™ Lipofectamine™ 2000 Transfection Reagent is a proprietary formulation for the transfection of nucleic acids (DNA and RNA) into eukaryotic cells Details can be found at: http://www. I use 24-well plates for transfections in triplicates. GFP Positive Expression Readings Using Flow Cytometry 293T cells were transfected by calcium chloride methods. Transfection P19 cells with Lipofectamine 2000 The same protocol can be used for 293T cells 1 Maintain P19 cells undifferentiated in MEM with 10 serum. 25 Aim: To establish a 293T cell line with stable expression of UBE2C and to investigate the effect of UBE2C overexpression on the proliferation of 293T cells. High efficiency, no toxicity(!), easy procedure, and low cost. html. 5 ml 293T cell suspension were mixed in 50 ml centrifuge tubes 293T cells were transfected by calcium chloride methods. 293Ts are not that adherent, so they will lift off of the plate if agitated. If this page load window, including pei for optimization plate purchaser notification this region of lipofectamine reagent outperforms other eukaryotic species. 5 µg pSARS-CoV2 Δ19. - Mix the DNA and the Lipofectamine 2000. After 48h of transduction, the lentiviral particles contained in the supernatant of 293T cells were harvested and then concentrated by passing through a 0. Then, lentivirus was produced in HEK293-FT packaging cells by co-transfecting the miR-BART9 All plasmid transfections were carried out using Lipofectamine Convenient protocol does not require removal of serum or culture medium and washing or changing of medium after introducing the reagent/DNA complex Cost effective Lipofector-EZ reagent is designed for the transfection of DNA or RNA into eukaryotic cells. For BAC DNA transfection in SLK cells, Lipofectamine 3000 was used according to the manufacturer's instructions. 5ul lipid complex to 50 ul plasmid mixture. After 24 hours, cells were collected and lysed in buffer containing 20 mmol/L Tris (pH 7. To prepare HEK 293T cells for reverse transfection: 1) 24 hours before needed, plate 10 x 106 cells in 10 ml media in a 10 cm dish. For 24 well plate: 1e5 cells/well in 0. After an overnight incubation, cells were lysed in protein lysis buffer (20 mM Tris-HCl pH 7. 293T were cotransfected with HA-SLC15A3 expression plasmids and Flag/DDK1-tagged cGAS, MAVS, STING, DDX41, and MyD88 using Lipofectamine 2000. Hong-Bing Shu of Wuhan University, and then amplified in our lab. We found that for optimal transfections, the total amount of plasmid DNA can reduced by 2. Using the 19-hour delayed shift of temperature from 37°C to *****Abbreviated protocol***** Surface area of 15 cm dish is 18 x greater than that of a 6-well dish. Catalog Number 11668-030 11668-027 11668-019 11668-500. Then, 4. Add 0. Co-Transfection of Plasmid DNA and siRNA Transfect plasmid DNA and siRNA at the same time using Lipofectamine® 2000 Reagent by adding 30 pmol (~0. Western blotting The cells were grown to 80~90% confluence and then Transfection, HEK293 or 293T adherent cells (CaPhos, Lipofectamine 2000) γ-Retroviral vector PG13-based stable producer cell line; Transfection, HEK 293T adherent cells (CaPhos) (35, 56) AAV Transfection, HEK293 (CaPhos) Foamy virus Transfection, 293T, (PEI) (58, 59) iCELLis™ (Pall) Adenoviral vector Infection 293T cells were transfected by calcium chloride methods.