please try PEI. its very straight forward,

Trying other transfection protocols (PEI, Lipofection, Effectene etc.) and/or waiting longer as suggested above may help.

But if you want to stay with FuGene HD and this vector you may also transfect in eg a 6-well plate (200.000 cells/well), wait 24-48 hours, check viability, and plate on 96 well plate only when a reasonable viability (more than 70%) is seen.

Or you make your own stably transfected cells: Do as above, but start treating with the selection antibiotic (presumably hygromycin or puromycin, your pGL4 vector card should state that), plate low numbers of cells (1000) into each well of a 96 well plate and check regularly in which proliferation is best. Replate the "best proliferators" check reporter gene activity, stock the best and use them.

If you don`t want to do that check the literature for the most successful transfection protocol for you cells and use that.

Good luck, Erik

THP-1 cells as most of the cell lines grown in suspension are generally difficult to transfect. You may try to use AMAXA Nucleofection system with a high efficiency transfection protocol for this particular cell type. We have been rather sucessful with this system, but be aware that it is rather expensive. Alternatively, treatment of THP-1 cells with PMA (post-tranfection)will increase the percentage of transgene expressing cells (this is at least the case for CMV-promoter containing plasmids). Also, you need to be aware that PMA is a potent inducer of differentiation and adherence in these cells, so it may affect the expression some other genes of importance, which could be important in your experimental setting.

Concerning Caco-2 I would use any standard transfection reagent. My experience is that it is better to transfect them under 30-40% confluency only with a bit higher amount of lipid/DNA complexes, but I would keep the standard lipid/DNA ratios. The reagent in this case was Fugene and monitoring of the transient transgene expression was 24-96h post-transfection.

Good luck!

Please check it whether electroporation can be applicable

If you haven't done so already, I would suggest trying a control cell line - an easy to transfect cell line like Hek293. Once you are confident that the plasmid and Fugene HD are both working properly, then I would try transfecting both THP1 and Caco2 before differentiating them. Once they differentiate, the cells might be harder to overexpress a 'foreign' protein.

We had that same problem with C2C12s. Once C2C12s become myotubes, they would not express a foreign gene - presumably due to the change in cell functions.

I concur with the others - try the different ratios of DNA:Fugene, longer expression times, different seeding densities. If possible, try nucleofection (e.g. Amaxa, Neon), Lipofectamine LTX, magnetic nanoparticle transfection... failing which, there is the dreaded Microinjection...

Hi Sridhar,

I too have been trying to transfect THP-1 using various transfection reagents. In our lab, we have been successful with lipofectamine and MACSfectin reagent (with about 10-14% transfection efficiency), which is similar to the efficiency received using electroporation. I am about to try using Fugene HD, so am pleased to see that there has been some success with the use of this with THP-1!

I agree with the above comments that an easy to transfect cell line, such as HEK-293T should be used to ensure your plasmid is functioning properly and then perhaps moving on to using GFP-1 to check transfection efficiency before finally using a luciferase reporter assay system. Additionally, we use the promega dual luciferase assay system to account for variability in transfection efficiency (the RLU is normalised to an internal control e.g. renilla or b-gal).

I hope this helps,

José

For THP-1 cells, I suggest DreamFect as shown by the paper attached, efficiency is quite good: Wenzel M., J Immunol. 2012 Jan 1;188(1):394-403. Epub 2011 Dec 2.

Hi, Sridhar!

I have transfected HEK293T cells using Fugene HD and for me this is the best option. I had many problems with transfection protocols… I tried Effectene, Lipofectamine 2000 and calcium phosphate. I plate 106 cells in a 60 mm dish and transfect 24 hours after this. I recover the supernatant 48 hour after the transfection and measure my protein production by ELISA. Many aspects could be involved… Are you checking in the microscopy your cells in each step (before and after transfection)?! I had attachment issues in the beginning and the rate transfection was so low. And I change the cells source (I took in another lab) and it was really better. I have never work with your cells, but my cell line transfect better after 5 passages and I don’t use antibiotics in the medium. I tried to transfect a few hour after plating and it was terrible. Did you try measure the NFKB production levels? Maybe there is some problem with the reporter. So, do you know this promega tool: http://www.promega.com/techserv/tools/FugeneHdTool/default.aspx

Don’t give up! You will learn a lot when overcome these obstacles!

Good Luck!

Taís Adelita

Dear Sridhar,

for your information a new paper just came out about transfecting siRNA in THP-1 cells with DreamFect. "RNAi-mediated silencing of MLL-AF9 reveals

leukemia-associated downstream targets and processes" Fleischmann et al molecular Cancer 2014. here is the link

http://www.ncbi.nlm.nih.gov/pubmed/24517546

Let me know if you need information about this reagent [email protected]

We are using our home-made APC targeting nanocarrier. Seems that we constantly get as high as 60 fold increase in the transfection compared to plasmid alone. I have asked the student to compare with Lipofectamin

Dear Sridhar,

I hope it is not too late to answer...;)

THP-1 cells are generally dificult to transfect, so do not have too high expectations. My point is that the transfection efficiency will be around 10-20%, when they grow in suspension. My advice is to transfect them first and then differentiate using any established protocol (e.g. PMA). This should help with expression of your luciferase. If you dont see expected values, you should consider trying different batch of cells.

Concerning Caco-2 cells it should be easier, just make sure you plate them sparsely (30-40% confluency) and wait 24-48h to see if your luciferase signal is optimally detected.

I hope it helps.

Good luck!

Kacper

Caco-2 and THP-1 should be simple to transfect if you have the appropriate reagents tailored to the cell lines (for Caco-2: https://altogen.com/product/caco-2-transfection-reagent-colon-carcinoma-cells/). THP-1 is a leukemia cell line, so lipofection won't be the best approach, and you may want to try electroporation that works for other leukemia cell lines (like Jurkat: https://altogen.com/product/jurkat-electroporation-kit-t-cell-leukemia-tib-152/). Your plasmid likely isn't the issue here, and it's probably best to find alternate transfection routes and see which one is most efficient.