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 lipofectamine 3000 transfection reagent. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. I use 24well plates for transfections in triplicates. Refer to the cell lines database for a list of cell types successfully. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Transitbrca transfection reagent research reagent products. Invitrogen lipofectamine 3000 transfection reagent fisher. Transfection of mcf 7 cells with lipofectamine 2000 and isolation of genomic dna abstract. B hela cells were transfected with luciferase encoding plasmid dna using either transit. It is possible to plate and transfect the cells on the same day, but cells must adhere to. Afcs procedure protocol pp00000182 version 1, 103103. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. We found that lipofectamine 3000 has much higher dna transfection efficiency than any other transfection reagents in mcf7 cells, the transfection efficiency reached 6080% 5kb gfp expression.
Cells from the mcf 7 breast cancer cell line are widely regarded as a near perfect model to examine the growth of malignant tumors. Altogen biosystems produces preoptimized highefficiency transfection reagent kits for the mcf 7 breast cancer cell line. Hi, im going to be doing some transfections of mcf 7 and mdamb231 cells, with a plasmid containg cat, and my gene of interest. Plate cells so they will be 7090% confluent at the time of transfection. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Add 6l of lipofectamine2000 from biostores and 100 l of. Lipofectamine 2000 reagent thermo fisher scientific.
Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Is it possible to use lipofectamine to transfect plasmid dna into. Prior to transfection, cells that had reached 8085% confluence in 6well culture dishes were added to 1500. Mcf7 cells and adipocytes were transfected with either the sirna sequence or the negative control mimic using lipofectamine 2000. I am looking for a protocol for plasmid transfection into mcf10a cells.
Which is the best protocol to transfect mcf7 cell line. Genscript recommends using lipofectamine 2000 for all transfections. Which is the best protocol to transfect mcf 7 cell line. Human adipocytes stimulate invasion of breast cancer mcf7. Cells from the mcf7 breast cancer cell line are widely regarded as a near perfect model to examine the growth of malignant tumors. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Is it possible to use lipofectamine to transfect plasmid dna into mcf7 cells using a 96 well method. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. As for mcf7 cells figure 10, lipofectamine 3000 demonstrated the highest transfection efficacy 58.
Altogen biosystems produces preoptimized highefficiency transfection reagent kits for the mcf7 breast cancer cell line. On the day of transfection just prior to preparing complexes, plate 1. This reference provides a recommended procedure to transfect plasmid dna into mcf7 human breast cancer cells atcc cat. Transfections were performed in 24well plates using 11. In biontex experiments gfp positivity increased to 90. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Similar to mcf7 cells, lipofectamine 3000, rnaimax and fugene showed. Systematic screening of commonly used commercial transfection. The following is a general protocol for use of dharmafect transfection. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages.
A sample protocol is listed here for transfection experiments performed in 6well plates. Please check for updated versions of this protocol. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. Hi, im going to be doing some transfections of mcf7 and mdamb231 cells, with a plasmid containg cat, and my gene of interest. Transfection of mcf7 cells with lipofectamine 2000 and isolation of genomic dna abstract. Transfection was measured by luciferase activity using a conventional assay.
Highest transfection efficiency in many cell types and formats e. Transfections were performed in 35 mm mattek dishes using 4 l of trans itbrca transfection reagent to deliver 2 g of dna 2. Cells should be plated on collagencoated dishes, as for normal propagation. Knockdown of lamin ac protein levels, using the same sirna, can also be accomplished by transfection using lipofectamine 2000. Lipofectamine 2000 transfection reagent dartmouth college. Although lipofectin showed the best safety profile with a cell viability of 92. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Lipofectamine 2000 transfection reagent from invitrogen. 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. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. We have lipofectamine 2000, and im just going through the protocol that is suggested by said manufacturer.
Transfection of mcf7 with lipofectamine 2000 maartje vogel. The breast cancer cell line hcc1806 and mcf7 cells grown to. Comparison of transfection efficiency of nonviral gene. Please check vansteensel for updated versions of this protocol. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Dharmafect volumes per well represent guidelines and need to be optimized. The activity of delivered protein is maintained so active enzymes and other biomolecules such as antibodies can be directly delivered to cells without the need to obtain and transfect an expression construct.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Mcf7 transfection reagent breast cancer, htb22 altogen. Transfex transfection of plasmid dna into mcf7 cells. Maintain p19 cells undifferentiated in mem with 10% serum 7. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. Invitrogen lipofectamine 3000 transfection reagent.
However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Fibroblast 3t3, cos 7 myoblast c2c12, l6 crl1458 hepatocyte hepg2, huh7 erythroleukemic cell k562. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetpei and expressfect. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Transfecting plasmid dna into mcf7 cells using lipofectamine. 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. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Polyethylenimine pei pei qbiogene, morgan irvine, ca and plasmid dna were each diluted with equal volumes of 150 mm nacl. Cell transfection mcf 7 cells in logarithmic growth phase were resuspended in 2 ml complete. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Transfecting plasmid dna into mcf7 cells using lipofectamine 3000 reagent. Maximal transfection efficiency in mc3t3e1, c3h10t12, hela, c2c12, hep g2, and hct116 was seen using fugene hd, in pt30, 4t1, and hekn was seen using arrestin, and in mcf7 was seen using jetpei.
As for mcf 7 cells figure 10, lipofectamine 3000 demonstrated the highest transfection efficacy 58. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. Puma gene transfection can enhance the sensitivity of epirubicininduced apoptosis of mcf 7 breast cancer cells c. Lipofectamine 2000 were purchased from gibco gaithersburg, md, usa. Superior transfection efficiency transitbrca outperforms lipofectamine 2000, in various breast cancer cell lines. Atcc achieved transfection efficiencies of 8590%, using the protocol described below. Protocol for lipofectamine transfection in nih3t3 cells transfection of mcf 7 with lipofectamine 2000 maartje vogel. Transfection was performed as described in protocol 2 with the following modifications. Transfection protocol lipofectamine 3000 transfection. To transfect cells with sirna, follow the protocol as. The transfection efficacy of lipofectamine 2000 was compromised by. Protocol for lipofectamine transfection in nih3t3 cells transfection of mcf7 with lipofectamine 2000 maartje vogel. Higher densities can lead to spontaneous differentiation. Advanced transfection with lipofectamine 2000 reagent.
Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene.
We found that lipofectamine 3000 has much higher dna transfection efficiency than any other transfection reagents in mcf 7 cells, the transfection efficiency reached 6080% 5kb gfp expression. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Puma gene transfection can enhance the sensitivity of. Invitrogen lipofectamine 2000 transfection reagent. 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. Maximal transfection efficiency in mc3t3e1, c3h10t12, hela, c2c12, hep g2, and hct116 was seen using fugene hd, in pt30, 4t1, and hekn was seen using arrestin, and in mcf 7 was seen using jetpei. Im trying to transfect mcf 7 with 2ug caspase3 gfp plasmid using lipofectamine2000 and the transfection efficiency is terribly low. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas.
Im trying to transfect mcf7 with 2ug caspase3 gfp plasmid using lipofectamine2000 and. The dnalipofectamine 2000 complexes were then added to each well containing cells and medium. Transitbrca transfection reagent was used to transfect plasmid dna encoding gfp into mcf7, mdamb231, mdamb453, mdamb468 and t47d breast cancer cell lines. Prewarm 50ml of optimum stored in cold room at 4c 10min. Laboratories and 5 ng of prltk using lipofectamine 2000. To transfect cells with sirna, follow the protocol as described for dna but. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Superior dna transfection of breast cancer cell types. Htb22 is an epithelial, breast adenocarcinoma cell line that was derived from a metastatic pleural effusion. Transfection protocol lipofectamine 3000 transfection reagent. Lipofectamine 2000 transfection protocol 10cm plates reagents. The following procedure is for simultaneous transfection and plating of raw 264. Have you thought of trying a reverse transfection protocol. Help on transfecting mcf7 and hct 116 cells transfection.
What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. Fibroblast 3t3, cos7 myoblast c2c12, l6 crl1458 hepatocyte hepg2, huh7 erythroleukemic cell k562. Lipofectamine 2000 dna transfection reagent protocol. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. For additional components required for the protocol see ordering table below. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Transpass p is a protein transfection reagent that can deliver intact functional proteins and peptides with high efficiency into live mammalian cells via endocytosis. The mechanism of lipofectamine 2000 mediated transmembrane.
1369 1432 1398 1234 1448 686 496 1534 1319 870 1325 1225 359 1527 35 997 821 378 790 491 790 57 863 1431 269 571 1409 712 418 196 2 1082