The overall efficiency depends on many factors, but using glioblastoma-designated transfection reagents (https://altogen.com/product/u87-transfection-reagent-glioblastoma-cells/) will help you get higher transfection efficiencies. It also depends on the size of your plasmid; larger plasmids require a complex condenser to shrink down the liposome-encapsulated cargo for cellular delivery. If you haven't had much cell death, then I would use higher concentrations of everything and see if it improves the results.

What is your lipofectamine2000 protocol? For how long time do you incubate your cells with lipofectamine? These cells (GL-261) are adherent cells?

GL-261 are adherent cells, originate from mouse glial cells. I follow their protocol (https://tools.thermofisher.com/content/sfs/manuals/Lipofectamine_2000_Reag_protocol.pdf) pretty much to the point, for the 6 well setup. I have tried all the different concentrations for lipofectamine and also tried different amounts of plasmid (producing eGFP). Never got over 5% transfection efficiency.

My point is about the time that you exposed cells to lipofectamine, because lipofectamine is toxic to the cells. When we do transfection with lipofectamine in adherent cells we incubate cells with lipofectamine for only 5 hours, and then we discard the optmem+ lipofectamine, Wash the cells with PBS for 3 times, add DMEM or the medium that you commonly use, after that we put the plate in Cell stove at 37°C for 24 hours, and finally we extract RNA after that.

I am going to transfect today. I will follow your recommendations.

Will let you know what the result was.

You only must put the optimem+lipofectamine+siRNA (or anti-miR) when your plate has a minimum of 65% of confluency and no more that 75%, because if you put the lipofectamine when the plate has near 100% (i.e. 90%) your cells will haven't space to increase after that, and it can be a problem.

I hope that it helps you.

Did you got make the transfection?

Yes, it did not make any difference. On the contrary: leaving the transfection mix on the cells for longer gave better results. Still never higher than 10%.

Hi Zijian,

We still have between 5% and 10% transfection efficiency at most, we've tried a lot of things including Lipofectamine2000 and Neon transfection system.

Let me know if you have better results.

Hi! I am facing the same problem. Could you improve the transfection efficiency?

Hi Raquel,

We are between 10-15% efficiency now, using the Neon transfection system.

Currently trying something new, which looks promosing, will let you know how it went.

What parameters are you using for the Neon transfection system? Also using the GLs and are trying to transfect them!

Dear Emiel,

When you tried the transfection with lipofectamine2000, did you use OptiMEM medium?

In my experiments, I used RNAimax also as transport agent instead the lipofectamine2000. For example, I named 2 tubes filled each with 250 uL of OptiMEM. In one of the tubes I put 1,3 uL antimiR at 75 nM concentration, and in the other tube I put 250 uL of RNAimax. After that, I mixed these two tubes (total volume of 526,3 uL) during 5 minutes. While I changed the medium in my flask for 4,5 mL of OptiMEM. Then I put the volume of the tube in the flask, and incubate this flask at 37ºC in 95% of CO2 and 5% of air during 5 hours. After that, I took the flask and discarded the medium, and washed the flask with PBS for 3 times. Then I put the normal medium in my flask, come back this flask to the incubator for more 24 hours. After that I did my experiments (cellular viability, RNA and miRNA extractions, proteins quantifying, immunofluorescence).

It worked for me. I mean, when I analyzed the mRNA of the target and the protein quantity, I have reached around 90% of efficiency, I know that because comparing the "wild type" cells with transfected cells, the results in my qPCR for this particular miRNA and its target, and the quantity by western blotting of the target protein almost reached 0.

I really hope that it helps you.

Pedro

Hi, I want to use this cell line. Can you please tell me how is going now with transfection? thank you. You anyway look smart.

@Evelina

We use the Mouse glial cells Neon protocol from Thermo (well documented on their website).

Hi,

In my opinion, GBM cells are simply hard to transfect with plasmids.

If I consider all the good feedbacks we have for siRNA transfection,

https://viromer-transfection.com/neurobiology-research/page/2

scroll down to see cell list (LN 229, LN18, NCH149, NCH82, T98G, Tu-132, Tu-140, U-251 MG, U-87 MG),

I have only one person who reported a sufficient efficiency for plasmid transfection.

see here, page 2, plasmid transfection in U-87 MG

https://viromer-transfection.com/wp-content/uploads/2015/09/U87-transfection-viromer.pdf

Even if our reagents for siRNA and for plasmid transfection are different in terms of structural chemistry, they have the same workflow. I assume that GBM cells can uptake transfection complex whatever the payload is. But what happens one into the cells is very different. Plasmid DNA needs to reach the nucleus for cellular action while siRNA not.

As an alternative, you should maybe consider using mRNA if you just need to overexpress a protein transiently.

https://viromer-transfection.com/mrna-transfection

If you want some more info about our Viromers, please let me know.

BR,

Sandra

The overall efficiency depends on many factors, but using glioblastoma-designated transfection reagents (https://altogen.com/product/u87-transfection-reagent-glioblastoma-cells/) will help you get higher transfection efficiencies. It also depends on the size of your plasmid; larger plasmids require a complex condenser to shrink down the liposome-encapsulated cargo for cellular delivery. If you haven't had much cell death, then I would use higher concentrations of everything and see if it improves the results.

I was able to get 50 % using lentivirus trasduction technique.

Simply you need to increase the MOI until you get a better Trasnduction.

Best

Mohammed