Thee two most important factors in choosing transfecting reagent are. Transfection protocol transfection components component cat. Essential 8 medium a1517001 vitronectin vtnn recombinant human protein, truncated a14700. Change cells to dmem, 10% fcs, no abx media prior to transfection 30 min by adding. Maintain p19 cells undifferentiated in mem with 10% serum 7. Add dna lipofectamine complexes 500 ul to the 2 ml of media on the cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Plate cells so they will be 6080% confluent at the time of transfection. I want to try to use hela cell and lipofectamine 2000 to do transfection. For the following protocol, we specify volumes for a 12well plate. This pathway includes deregulated genes coding for heat shock proteins.
I use 24well plates for transfections in triplicates. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Dharmafect reagents are effective across a broader range of experimental conditions when compared to lipofectamine 2000 invitrogen. Lipofectamine transfection reagent is one of our firstgeneration, cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells. Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine 2000 transfection reagent dartmouth college. Dharmafect sirna transfection reagents ensure effective delivery across a broad range of cell densities and lipid volumes with minimal effect on cell viability. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. For each transfection sample, prepare oligomer lipofectamine 2000 complexes as follows. Thermo scientific nunc 24well cell culture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. Liposomebased dna carriers may induce cellular stress. Before seeding cells for virus package, make sure hek293t cells are in rapid replication state by growth cells to approximately 75%80% confluence in a 10 cm cell culture dish and passaging at 1. Lentiviral transduction is one of the most effective delivery system for stable gene expression.
Prior to use of lipofection on an alternative cell line, we strongly recommend doing. In this study, sw480 cells were transfected with plasmid containing green. Invitrogen lipofectamine 3000 invitrogen lipofectamine 3000 transfection reagent 1. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. The reagent provides high transfection efficiency in many cells types and formats. Purify the linear fragment using any appropriate method. To transfect cells with sirna, follow the protocol as. It consistently produces high transfection efficiency and high protein overexpression. The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure.
Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. Lipofectamine transfection reagent is a trusted, value reagent with many years of cited use in thousands of publications and with many cell lines. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Cells should be plated on collagencoated dishes, as for normal propagation. Optimized conditions for successful transfection of human. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Plate cells in a 35mm dish at least a day before transfection b. Transfecting a human neuroblastoma cell line with monster.
Transfection protocol lipofectamine stem transfection reagent transfection of pluripotent stem cells with lipofectamine stem transfection reagent in essential 8 medium psc growth medium, passaging reagents, and complexation medium component cat. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate.
Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Plate cells for transfection only 1 day before the experiment. Dilute 20 pmol stealth rnai or sirna oligomer in 50 l gibco optimem i. Comparative transfection efficiency of jetprime versus its main competitor. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells. During functional studies on the rat stressinducible hspa1b hsp70. Lipofectamine transfection of c2c12 cells bridges lab. Transfecting cells at a lower density allows a longer interval between transfection and assay time, and minimizes the loss of cell viability due to cell overgrowth. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. A highly efficient method of transfecting hep g2 cells. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. The lenti293t cell line has been tested as a suitable cell line for lentiviral production due to high transfection efficiency, high level of protein yield and high titer of lentiviral production.
Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Other reagents did not demonstrate effective transfection to this cell line. Cell transfection protocol for lipofectamine 2000 transfection of a 35 mm dish of 80% confluent cho cells. I want to try to use hela cell and lipofectamine 2000 to. Optimizing transfection lipofectamine 2000 cd reagent. After 4h aspirate media and refeed cells with normal media. Transfection of pluripotent stem cells with lipofectamine. Different cell confluencies, dnareagent ratios and total transfection volumes were optimized for two chemical reagents including jetpei and lipofectamine 2000. Seeding cells for transfection the day before transfection, dissociate cells that are 8090% confluent in a t75 flask.
Dna is enclosed within the liposomes and the complexes fuse with the host cell membrane for dna entry into the cytoplasm and eventu ally into the nucleus. Product description lipofectamine2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Evaluation of transfection methods for transient gene. Lipofectamine 2000 transfection protocol 10cm plates reagents. Transfection protocol lipofectamine stem transfection reagent transfection of pluripotent stem cells with lipofectamine stem reagent in mtesr1 medium on geltrex matrix psc growth medium, passaging reagents, and complexation medium component cat. Plate cells so they will be 7090% confluent at the time of transfection. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Highest transfection efficiency in many cell types and formats. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount.
Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Mammalian cell transfection can be done utilizing a variety of techniques. Lipofectamine 2000 transfection reagent from invitrogen. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. The animal originfree formulation ensures that mammalian cell culture and bioproduction processes are free of animalderived materials. It can be used for transfection of adherent or suspension cells with dna or rna including stealth rnai and sirna and can be used for either transient transfections or to create stable cell lines. The bioluminescence system luciferase reporter assay system is widely used to study gene expression, signal.
63 1233 559 1297 878 1542 895 1246 1034 803 952 801 1602 388 132 448 576 857 1385 738 741 254 1532 469 8 489 540 768 1529 455 875 1273 1639 799 491 1314 1431 694 1285 1104 1238 707 914 637 316 137 1048