For anyone who still is interested: Finally, we have cracked it. We are using Nepa21 electroporator (Nepagene) and are getting close to 70% transfection efficiency in C2C12 myotubes. Conditions are: Transfection medium: Optimem.  Poring pulse: two Pulses@ 300V ( length 7.5ms) with interval of 50ms. Decay rate 10.  Transfer pulse: 5 pulses @ 30V (length 50ms) interval 50ms, decay rate 40, Polarity +/- .

Dear Sabyasachi,

I would recommend you two transfection reagents that were demonstrated to work efficiently either for siRNA and DNA transfection, respectively Dreamfect and DreamFect Gold.

• for DNA/DreamFect Gold: .Vafiadaki E., FEBS J. 2014 May 27. doi: 10.1111/febs.12859.
• for siRNA/DreamFect: Tajika Y., Acta Histochem Cytochem. 2010 Aug 27;43(4):107-14.

for DNA transfection, the protocol is really simple,

would you like to try these reagent please contact me at: [email protected]

good luck in your experiment,

best regards,

Cedric

http://www.ncbi.nlm.nih.gov/pubmed/24860983

http://www.ncbi.nlm.nih.gov/pubmed/20824121

Dear Cedric,

Thanks for the response. However, it appears none of the papers you cited actually transfected plasmids in myotubes. The FEBS J paper transfected myoblasts and allowed them to differentiate(I get good enough result with Lipofectamine LTX there). The Acta Histochem Cytochem paper used CombiMag, or SilenceMag to perform knockdown. I in fact do get nice results for knockdown using siRNAs from Dharmacon and Dharmafect I . I will appreciate if you can give me any reference for plasmid transfection in C2c12 myotubes.

Best regards

Sabyasachi

Dear Sabyasachi,

I am really sorry for the confusion between myoblasts and myotubes in the publications; even the publication on primary myoblasts (ref in following link) transfected with DreamFect Gold do not mention  myotube transfection. I am thus afraid not to have an article on myotube transfection to recommend you.

Maybe you could be interested in testing this reagent anyway ? just tell me

I do hope you will succeed in your experiment;

all the best,

Cedric

http://www.ncbi.nlm.nih.gov/pubmed/20884321

Dear Cedric,

Thanks again and yes, no harm in testing-- I would like to test Dreamfect.

Best Regards

Sabyasachi

Hey poet!  good question.  Why not use a gene-gun from BioRad (#165-2431)?.  See Antolik C  Sci STKE 2003 Jul 22, 2003 (192) PL 11.   They adapted a system previously used to transfect plant cells to myotubes from chicken and C2C12 myotubes.  Anyone that transfects  plant cells might help.

Hello Sabyasachi, 

I would suggest the modified Lipofectamine 2000 (Invitrogen) protocol described by Mercer et al., 2005. (http://www.jbc.org/content/280/27/25788.full).

This protocol was recommended by researchers doing routine C2C12 transient transfections. I have used it in MEFs and it also improved the transfection efficiency significantly. Hope it helps!

Dear John,

Thanks but the gene gun is way too expensive (will like to get one someday) for me. I was looking for a cheaper way.

Dear Indra,

Thanks but the paper you refer to is about myoblast transfection. I use Lipofectamine LTX to transfect myoblasts and no problem there.

Sincerely,

sabyasachi

Dear Sabyashi, 

I'm sorry I misundesrtood you wanted a protocol for C2C12. In that JBC paper they describe in detail the modification to transfect C2C12 and they improve efficiency from 20% to 80%. 

Best wishes,

Indra

I would be very surprised if you found examples in the literature of successful transfections of fully differentiated C2C12 myotubes. In our hands, nothing has worked and we turned to adenovirus, which worked well. I also recall a paper using electroporation that claimed positive results.

Dear Indra,

Thanks. The paper was a good read anyway.

Dear Gonzalo,

Thanks. I guess adenovirus might be the only option (although I don't have the virus facility at my disposal; and thus will try Dreamfect as Cedric suggested). Re: Electroporation, doesn't it need the cells to be trysinized? Can you trypsinize the myotubes and replate?

No, you electroporate directly on the coverslips (see http://www.sciencedirect.com/science/article/pii/S0014482703000971), but I don't think many have used this method. For Adenoviruses, we followed the protocol using the gateway vector from invitrogen (Virapower), no need to for sophisticated facilities there, just the vectors and 393A cells. Good luck!

Dear Gonzalo,

Thanks a lot. Will give it a try.

Let's have a look there https://www.ssl-id.de/www.lipocalyx.de/myocyte-transfection/?force_sid=6g54lhrbl4kfimvuko7vkoah97

;-)

Tell me if you are interested...

Cheers,

Sandra

Dear Sandra,

Thanks. Heard about viromers before but not in the context of C2c12 myotubes. Will gladly give it a try. Can I get a sample?

Regards

Sabyasachi

Thanks for your message. I answered you on your CDRI email... hope it works. Best, Sandra.

Thanks for the mail Sandra. Replied.

Regards

Sabyasachi

 For anyone who still is interested: Finally, we have cracked it. We are using Nepa21 electroporator (Nepagene) and are getting close to 70% transfection efficiency in C2C12 myotubes. Conditions are: Transfection medium: Optimem.  Poring pulse: two Pulses@ 300V ( length 7.5ms) with interval of 50ms. Decay rate 10.  Transfer pulse: 5 pulses @ 30V (length 50ms) interval 50ms, decay rate 40, Polarity +/- .

Hi Sabyasachi, Would you mind sending me your protocol? Do you use the coverslip method described in the paper above? Thanks

Dear Troy, with Nepagene we directly do it in a 6 well plate. They have an electrode that you can dip in the well. 

We are using Nepa21 electroporator (Nepagene) and are getting close to 70% transfection efficiency in C2C12 myotubes. Conditions are: Transfection medium: Optimem.  Poring pulse: two Pulses@ 300V ( length 7.5ms) with interval of 50ms. Decay rate 10.  Transfer pulse: 5 pulses @ 30V (length 50ms) interval 50ms, decay rate 40, Polarity +/- .

Can you suggest a protocol for transfecting C2C12 myotubes (not myoblasts)? - ResearchGate. Available from: https://www.researchgate.net/post/Can_you_suggest_a_protocol_for_transfecting_C2C12_myotubes_not_myoblasts#57a6c7c1217e20f76b68d921 [accessed Sep 14, 2016].

Dear Sabyasachi,

Do you have any theories on why it is so difficult to transfect differenciated C2C12 cells?

Hello Luisa,

My theory - maybe wrong but... - is that differentiated cells do not divide enough. Even if you manage to transfer DNA into the cytosol (by electroporation or by using a chemical transfectant), the nucleus pores need to be opened to enable the delivered DNA reaching the cell machinery for transcription into mRNA. An alternative for non-dividing cells is to work directly with mRNA (if your goal is to overexpress a protein transiently). mRNA is directly active once into the cytosol and instructs cells for protein production as early as 2-4h after delivery.

For more details, you can go here

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

At your disposal if you need more info about our technology.

Bests, Sandra

Dear Sabyasachi Sanyal ,

We will be testing the NEPA21 system for C2C12 myotubes. Could you provide some more details to your protocol? How much DNA to use per well on 6-well plate, what kind of volumes, when to change media after transfection etc.? Thanks in advance!

Best regards,

Jaakko Sarparanta

Hey Jaakko,

It looks like Sabyasachi was using a protocol similar to this paper: Article UBE2E1 Is Preferentially Expressed in the Cytoplasm of Slow-...

0.46ug DNA / uL in optimem. 300ul optimem / well of a 24 well plate. After electroporation, switch to growth media without antibiotics for 1.5 hrs, then switch to normal differentiation media.

Hi all,

Very helpful information, thank you!

I was wondering, how do you quantify efficiency of transfection in myotubes?

I am thinking of how to do it, but I can't think of a way to count the myotubes, because they are elongated, fused and with many nuclei. Do you count them manually?

The only way I can think of at the moment is to do DAPI staining and calculate the ratio GFP cells/DAPI nuclei and compare it between different conditions to figure out what is the best transfection protocol, but I don't know how to calculate it as a percentage. Also, with this ratio 1 GFP+ cell could have many DAPI+ nuclei, in the case of multinucleated myotubes, so it is not very reliable.

Thank you!