...DNA into HUVEC Cells Using Lipofectamine LTX Reagent |...

Home Technical Reference Library Transfection Protocols Transfecting Plasmid DNA into HUVEC Cells Using Lipofectamine LTX ReagentTransfecting Plasmid DNA into HUVEC Cells Using Lipofectamine LTX Reagent展开导航‹ See all transfection protocols Quicklinks IntroductionMaterials Ordering InformationTransfection of HUVEC CellsScaling Up or Down Transfections Related Product Information Transfection Selection Mammalian Cell CultureRNAi IntroductionLipofectamine® LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. This reference provides a recommended procedure to transfect plasmid DNA into HUVEC Human Umbilical Vein Endothelial primary isolates (passage 0, Cambrex, East Rutherford, NJ) using Lipofectamine® LTX Reagent (Cat. No. 15338-100). Important Guidelines for Transfection Follow these important guidelines when transfecting DNA into HUVEC cells using Lipofectamine® LTX Reagent:The addition of antibiotics to media during transfection may result in cell death, and has not been tested for HUVEC cells. If you wish to use antibiotics during transfection, test your conditions thoroughly.Maintain the same seeding conditions between experiments. Use low-passage cells; make sure that cell are healthy and greater than 90% viable before transfection.Transfection can be performed both in the presence or absence of serum. Test serum-free media for compatibility with Lipofectamine® LTX Reagent.Using PLUS™ Reagent (Cat. No. 11514-015) enhances transfection performance in HUVEC cells.We recommend Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute the DNA and Lipofectamine® LTX Reagent before complexing.Visit www.lifetechnologies.com/transfection or contact Technical Service for other specialized transfection protocols (including cell-type specific advice on use of PLUS™ Reagent and antibiotics, and a protocol for vector-based RNAi).Lipofectamine® LTX Reagent performs well with vector-based RNAi experiments. For siRNA and Stealth™ RNAi transfections, we recommend Lipofectamine® RNAiMAX (Cat. No. 13778-075). Go towww.lifetechnologies.com/RNAi or contact Technical Service for more information.Materials Needed Have the following reagents on hand before beginning:HUVEC cells maintained in Endothelial Cell Medium-2 (Clonetics Cat. No. CC-3156). Grow cells at 37°C with 5% CO2.Plasmid DNA of interest (100 ng/μl or higher)Lipofectamine® LTX Reagent (store at +4°C until use), and PLUS™ Reagent (if desired; store at 4°C)Opti-MEM® I Reduced Serum MediumAppropriate tissue culture plates and suppliesTOP15338100,15338500 Transfection of HUVEC Cells Use this procedure to transfect plasmid DNA into HUVEC cells in a 24-well format (for other formats, see Scaling Up or Down Transfections, below). All amounts and volumes are given on a per well basis. The day before transfection, trypsinize and count the cells. Plate 4 x 104 cells per well in 0.5 ml of complete growth medium. Cell density should be 50~80% confluent on the day of transfection. For each well of cells to be transfected, dilute 0.5 μg of DNA into 100 μl of Opti-MEM® I Reduced Serum Medium without serum. If using PLUS™ Reagent: Mix PLUS™ Reagent gently before use, then add 0.5 μl PLUS™ Reagent (a 1:1 ratio to DNA) directly to the diluted DNA. Mix gently and incubate for 5-15 minutes at room temperature. For each well of cells, dilute 0.75-2.75 μl of Lipofectamine® LTX into the above diluted DNA solution, mix gently and incubate for 25 minutes at room temperature to form DNA-Lipofectamine® LTX complexes. Remove growth medium from cells and replace with 0.5 ml of complete growth medium. Add 100 μl of the DNA-Lipofectamine® LTX complexes directly to each well containing cells and mix gently by rocking the plate back and forth. Complexes do not have to be removed following transfection. Incubate the cells at 37°C in a CO2 incubator for 18-24 hours post-transfection before assaying for transgene expression. TOP Scaling Up or Down Transfections To transfect HUVEC cells in different tissue culture formats, vary the amounts of Lipofectamine® LTX Reagent, DNA, cells, medium and PLUS™ Reagent used in proportion to the relative surface area, as shown in the table (amounts given on a per well basis).Culture vessel Surfacearea perwell1 Volume plating medium Cells per well Volumedilutionmedium2 DNA Lipofectamine®LTX ReagentPLUS™Reagent96-well0.3 cm2100 μl8 x 10320 μl100 ng0.25 - 0.3 μl0.1 μl48-well1 cm2200 μl2 x 10440 μl200 ng0.5 - 0.6 μl0.2 μl24-well2 cm2500 μl4 x 104100 μl500 ng1.25 - 1.5 μl0.5 μl12-well4 cm21 ml8 x 104200 μl1 μg2.5 - 3.0 μl1.0 μl6-well10 cm2 2 ml2 x 105500 μl2.5 μg6.25 - 7.5 μl2.5 μl1 Surface areas may vary depending on the manufacturer. 2 If the volume of Lipofectamine® LTX Reagent is too small to dispense accurately, and you cannot pool dilutions, predilute Lipofectamine® LTX Reagent 10-fold in Opti-MEM® I Reduced Serum Medium, and dispense a 10-fold higher amount (should be at least 1.0 μl per well). Discard any unused diluted Lipofectamine® LTX Reagent. TOP25-0938W9 Jun 2006