Higher densities can lead to spontaneous differentiation. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. The day before transfection, trypsinize and count the cells. Lipofectamine transfection of c2c12 cells bridges lab protocols.
Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Trypsinize hepg2 cells with tryple dissociation reagent for 1015 min in a 37c. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Transfection of mcf 7 cells use this procedure to transfect plasmid dna into mcf 7 cells in a 24well format for other formats, see scaling up or down transfections, below. Invitrogen lipofectamine 2000 transfection reagent. Using four different transfection reagents fugene hd, lipofectamine 2000, effectene, and lipofectamine ltx with plus reagent, jacobsen et al. Gibco dmem with glutamax supplement a3635201 10% gibco fbs a3160401 1. I use 24well plates for transfections in triplicates. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. A reverse transfection protocol was used to transfect mcf7 and hepg2 cells. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. All amounts and volumes are given on a per well basis.
Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. 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. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. For your convenience, the essential components of this protocol are now available in the gibco lung cancer starter kit. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Prior to transfection, cells that had reached 8085% confluence in 6well culture dishes were added to 1500. As for mcf 7 cells figure 10, lipofectamine 3000 demonstrated the highest transfection efficacy 58. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Transfecting plasmid dna into mcf7 cells using lipofectamine 3000 reagent. From the luciferase activities and consistencies of data using different transfection reagents including genejammer, effectine, wellfect, lipofectamine, what i can say from the data i observed, the efficiency of transfection is much more better in hct116 cells. Generally need to have 2 g of dna for each tranfection. Transfecting plasmid dna into mcf7 cells using lipofectamine. Prewarm 50ml of optimum stored in cold room at 4c 10min. Luciferase expression was compared at 24 hours posttransfection using a standard luciferase assay. Transfection, nucleofection, rnai, sirna, breast cancer, glioma, cancer stem. Maintain p19 cells undifferentiated in mem with 10% serum 7. However, significant toxicity from lipofectamine 2000 was also.
Ordering information gibco breast cancer starter kit. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Please check vansteensel for updated versions of this protocol. In this case, the cas9 rnps and lipofectamine crisprmax reagent were prepared in two separate tubes as described. We have lipofectamine 2000, and im just going through the protocol that is suggested by said manufacturer. Im trying to transfect mcf7 with 2ug caspase3 gfp plasmid using lipofectamine2000 and the transfection efficiency is terribly low. 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. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Lipofectamine 2000 reagent thermo fisher scientific. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. P ratio gave 70fold higher luciferase expression as assessed by rlu.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs.
Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells. Lipofectamine transfection of c2c12 cells bridges lab. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. This reference provides a recommended procedure to transfect plasmid dna into mcf7 human breast cancer cells atcc cat. Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Help on transfecting mcf7 and hct 116 cells transfection. Transfection protocol lipofectamine 3000 transfection reagent. Altogen biosystems produces preoptimized highefficiency transfection reagent kits for the mcf 7 breast cancer cell line. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Protocol for lipofectamine transfection in nih3t3 cells transfection of mcf 7 with lipofectamine 2000 maartje vogel. I am planning to use the same reagent in mcf7 cells in recent.
Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Mcf7 cells and adipocytes were transfected with either the sirna sequence or the negative control mimic using lipofectamine 2000. Protocol for lipofectamine transfection in nih3t3 cells transfection of mcf7 with lipofectamine 2000 maartje vogel. Transfection of mcf7 cells with lipofectamine 2000 and isolation of genomic dna abstract. The intracellular trafficking mechanism of lipofectamine. Lipofectamine 2000 transfection protocol 10cm plates reagents. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Gibco hams f12 nutrient mix 11765054 5% horse serum 1 mm gibco sodium pyruvate 160070 25 mm gibco hepes 15630106 proper culture techniques and procedures are an essential. Stealthsirna transfection protocol lipofectamine 2000. The vector contained an ofp reporter gene and was transfected with lipofectamine 2000 or lipofectamine 3000 reagent into a u2os and b hepg2 cell lines. Transfection of human breast adenocarcinoma mcf7 cells with fugene hd, lipofectamine 2000 and viromer red viromer outperforms major competitors good cell viability vr01lb00, vr01lb01 mcf 7. Lipofectamine 2000 right panel around 5% efficiency. The transitx2 dynamic delivery system mirus bio and lipofectamine 2000 transfection reagent thermo fisher scientific were used to transfect plasmid dna encoding luciferase into 41 different cell types at three reagenttodna ratios. For your convenience, the essential components of this protocol are now available in the gibco breast cancer starter kit.
Although lipofectin showed the best safety profile with a cell viability of 92. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Has anyone used lipofectamine 3000 for plasmid transfection. Similar to mcf7 cells, lipofectamine 3000, rnaimax and fugene showed the highest. Optimization of sirna transfection for breast and glioma. Transfection of mcf7 with lipofectamine 2000 maartje vogel.
For additional information on scaling your transfection reaction, see page 4. This protocol is for transfecting plasmid dna into mammalian cells. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. I am getting shitty transfection efficiency using effectene and geneporter 2 transfection reagent.
Transfection of cells should be performed only between passages 5 and 25 postthaw. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. If designing an experiment that involves transfection, ensure that setup coincides with a cell passage. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Transfection of human breast adenocarcinoma mcf 7 cells with fugene hd, lipofectamine 2000 and viromer red viromer outperforms major competitors good cell viability vr01lb00, vr01lb01 mcf7.
Hi, im going to be doing some transfections of mcf7 and mdamb231 cells, with a plasmid containg cat, and my gene of interest. A sample protocol is listed here for transfection experiments performed in 6well plates. Highest transfection efficiency in many cell types and formats e. Herein, we report the transfection efficiency and toxicity. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Optimization of transfection methods for huh7 and vero. Transfection of mcf 7 cells with lipofectamine 2000 and isolation of genomic dna abstract. Transfection protocol on the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using invitrogen lipofectamine 3000 transfection reagent. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Optimization of sirna transfection for breast and glioma cancer cell cultures rna silencing technology and methods based thereupon are important in todays medical biotechnology.
This protocol is well adjusted for cancer cell lines. Which is the best protocol to transfect mcf 7 cell line. Laboratories and 5 ng of prltk using lipofectamine 2000. As for mcf7 cells figure 10, lipofectamine 3000 demonstrated the highest transfection efficacy 58. Add 6l of lipofectamine2000 from biostores and 100 l of. Cells from the mcf 7 breast cancer cell line are widely regarded as a near perfect model to examine the growth of malignant tumors. Small interfering rna sirna highthroughput screening has a variety of. Im trying to transfect mcf 7 with 2ug caspase3 gfp plasmid using lipofectamine2000 and the transfection efficiency is terribly low. A comparison showing transfection efficiency of polyjet reagent vs. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. I didnt use it in mcf7 cells, but i used it in 293t cells. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. The transfection efficacy of lipofectamine 2000 was compromised by.
Systematic screening of commonly used commercial transfection. Silentfect biorad, hercules, ca is a transfection reagent commonly used at vtt turku at the moment. I didnt use it in mcf 7 cells, but i used it in 293t cells. Transfection protocol lipofectamine 3000 transfection reagent mcf7 breast cancer cells complete growth medium component cat. It is possible to plate and transfect the cells on the same day, but. The transfection protocol for lipofectamine 3000 reagent was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Lipofectamine 2000 thermo fisher scientific and 25 nm sirna according to each manufacturers protocol. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Despite this, a satisfactory mechanismbased explanation of their. Improved delivery of cas9 proteingrna complexes using. Plate cells for transfection only 1 day before the experiment.
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