I'm trying to transfect 4T1 cells with Lipofectamine and it doesn't work at all. I did a transfection efficiency assay with a RFP plasmid, and only 2% of my cells were effectively transfected. Has anybody already had this problem?
I have had problems transfecting several cancer cell lines, but not 4T1s. Invitrogen has some protocols on their website for difficult to transfect cell lines. Really, I have found the most helpful thing to do is set up a matrix in a 12w plate with varied amounts of lipofectamine and [DNA]. I think Invitrogen also makes some additive to help transfection efficiencies. Other general things to help - try varying the hours between seeding and transfecting. Generally 12-16 hours is good and the confluency (around 70%). And add the DNA and the transfection reagent directly to media, don't drip it down the plastic tube. I don't know if this is helpful or not, but good luck!
Invitrogen also recommends reverse transfection for improved delivery of plasmid/siRNA. You could try this: prepare transfection cocktail in the wells and add appropriate number of cells directly to well. As cells are still in suspension there is more cell surface area for the Lipofectamine to interact with, hope this helps and good luc with your experiment.
I can recommend the reverse transfection. It worked and was 100% succsesful ;) with T47D cells (breast cancer)
Thanks! So for reverse transfection, should I use more cells than for classic transfection ?
it is known that malignant tumors are sinks for low density lipoproteins which means up-regulated receptors for LDL, VLDL, and IDL. All of which bind to nucleic acids. LDL and VLDL are also known to bind DNA in lupus and Hepatitis C virus RNA. We use purified LDL in transfection of cells and we have seen that some cells are rapidly transfected while others are not. we usually prime the cells by incubating in an FBS-free medium for 3 - 4 hrs, this depends on the cell type. we have also primed cells by incubating overnite in DMEM-3%FBS-metastatin with similar outcome. we have developed a rapid method for isolating LDL from most plasmas. hope this helps.
I have used neon transfection (life technologies) for difficult to transfect cell lines. I have not used 4T1 cells personally. As some people have mentioned earlier, I have also found that density of cells at the time of transfection and surface area exposed to the transfection reagents are important variables that influence the efficiency of transfection and both vary from cell line to cell line.
I had done the same work on 4t1 cells.I transfected with pEGFPN1 plasmid containing EGFP as monitoring marker by lipofectamin plus reagent .The first assy when I was done by FlowCytometry for GFP detection, I had 10% positive cells, but because I needed stable cell line, I used selection marker G418 and step by step outcomes became better and finally I reached 98% positive cell clone with good MFI for GFP expression.so I think if you want to increase your positive cell(if stable cell line you want) you should select your cells by selective marker like Antibiotics and if you want transiant expression its better to set different amount of lipofectamin and plasmid to reach the best
I also suggest PolyEthylenimine tranfection protocol that was published by nature protocol2012 as altarnative method
By best
4T1 cells are very stubborn. Effective gene expression in this cell line can be achieved through gene transduction using retro- or lenti-viral mammalian expression constructs. While direct transfection can be achieved, you will commonly see low efficiency and very short term expression (
For plasmid transfection of my breast cancer cell lines I use Viromer RED (from Lipocalyx).
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