Working with the C2C12 cell line

C2C12 is an immortalized mouse myoblast cell line. The cells readily proliferate in high-serum conditions, and differentiate and fuse in low-serum conditions. While this cell line is a very useful tool to study aspects of myogenesis, metabolism and muscle biology, there are a number of important limitations that should be taken into account. Personally I prefer to work with either mouse primary myoblasts or single fibers (with satellite cells attached), both of these methods are described in detail in a separate post.  Here are some basic methods for dealing with C2C12 cells:

Proliferation of C2C12 cells 

  • Heat growth media (GM – DMEM (25mM glucose), 20% fetal bovine serum, and penicillin/streptomycin) to 37 degrees Celsius
  • Dilute cells to required density (I like to plate 30-40%, which is ~100k cells/mL) in warm growth media in a 50mL falcon tube
  • Plate as required (directly on tissue culture dish), and incubate at 37 degrees Celsius in 5% CO2
  • Growth media should be replaced every two-days, and cells should not be allowed to reach >70% confluency, before splitting (as they will differentiate)

Splitting and replating C2C12 cells

  • Heat GM (see above) and 0.25% Trypsin-EDTA to 37 degrees Celsius
  • Remove cells to be split from incubator and aspirate off any media
  • Wash plate twice with PBS (at room temperature), and aspirate off all PBS
  • Add warm Trypsin to plate (I use 1mL for a 100mm dish, and 0.2mL for a 35mm dish), and return plate to 37 degree incubator for 3-5minutes
  • Cells should now float freely around in the dish, add GM to the dish to inhibit the trypsin
  • Move cell suspension to a 15mL falcon tube and centrifuge at a low speed for 5mins
  • Resuspend the cells in a small volume of GM, count cell number on haemocytometer (or automated cell counter)
  • Follow protocol above for proliferation of C2C12 cells

Differentiation of C2C12 cells

  • Heat differentiation media (DM – DMEM (25mM glucose), 2% horse serum, and penicillin/streptomycin) to 37 degrees Celsius
  • Cells to be differentiated should be ~90-100% confluent
  • Wash plate twice with PBS (at room temperature), and aspirate off all PBS
  • Add warm DM to plate and return plates to incubator at 37 degrees Celsius and 5% CO2
  • Differentiation media should be changed every 24hours by removal of 75-90% of previous media, and then addition of new (warmed to 37 degrees) DM back to original volume
  • Myogenin should be detected by immunofluorescence or western immunoblot within 24 hours of incubation in DM (see image below). Myosin heavy chain should be detectable within 48-72 hours
  • The rate of differentiation can be increased if DM is supplemented with insulin/transferrin/selenium (ITS), NOTE: This will activate the insulin signalling pathway

Figure – C2C12 differentiation timecourse. C2C12 myoblast cells will begin to express myogenin upon reaching 100% confluency, When changed to differentiation media, the cells will rapidly upregulate myogenin, and differentiate to form myotubes,


165 thoughts on “Working with the C2C12 cell line

  1. RRW

    This is very helpful, do you have a protocol for performing IFA on differentiated C2C12 myotubes?
    The main problem that I am experiencing is that after 4 days differentiating, and then after 2 more days of transduction (I am using lentiviral mediated expression of my protein of interest), the cells begin to wash off the collagen coated coverslips that I am using. Do you have any suggestions? Thank you

    1. The thing about C2C12 cells is that once they reach a certain stage of differentiation, they will begin to contract and pull up off the collagen. Is it essential for you to differentiate for 4 days? If not, you could differentiate for 3days and then transduce your cells.

    2. Ana

      We are trying to transduce myotubes with lentivirus but they are not getting transduced. Could you detail your lentiviral transduction protocol and which is your efficiency?
      Thank you very much

  2. KK


    What if they don’t differentiate? I came across such problem recently. Previously I performed differentiation many times, but since I moved to a new lab, my C2C12 differentiate less efficiently. I tried to use an aliquot of cells from another lab, but the problem didn’t disappear. I would be grateful for any ideas which factor can be the critical one.

    1. KK,

      There are several things for you to consider, are these C2C12 cells from a later passage number (passage number >15 will have reduced proliferation/differentiation), if the problem is consistent across several aliquots you may want to check for contamination issues, or whether your incubator may not be keeping temp/CO2

      1. KK

        Thanks for advices.
        Problem was horse serum. Heat inactivated works much better. I didn’t expect that because previously it worked with non-heat inactivated serum and I didn’t have any problems. Apparently serum to serum difference still is an issue nowadays.

        @Manuela: Nice to know.


  3. FD

    Until what passage number do you differentiate the C2C12 cells? I experienced that at higher passage numbers the cells don’t differentiate that well anymore.

    1. FD,

      If you keep your cells below passage 15 from when you receive them from ATCC then you should be fine (best thing to do is freeze 100+ aliquots at passage 2-3, and then replenish as needed). I try to keep my stocks at <6-8 passages.

    2. KK

      I always tried to work with low passage number because it is a “common advice” but in ATCC they also need to split them…
      Don’t you think that it doesn’t matter so much as long as you don’t let them grow over-confluent?

      1. FD

        At high passage numbers the cells do not differentiate that good anymore! But if you don’t differentiate them, I think you can grow them to higher passage numbers.

  4. Petey

    After differentiating the myoblasts into myotubes, how would you remove the non-differentiated myoblasts in order to study only myotubes?

    1. Hi Petey,

      There are a number of papers describing a protocol to do exactly what you are looking for. Generally people are interested in the revers (ie. focusing on the undifferentiated “reserve cells”). If you search for “C2C12 reserve cells” you should be able to find a protocol pretty easily. Basically, you are going to use a light trypsinization protocol (ie. 1:1-5 in PBS) and watch carefully under a microscope until the myoblasts come loose. You should then be able to wash the myoblasts off, leaving you with a pure myotube sample.

  5. Sarah

    Hi, I’m new to cell culture. I will be using C2C12 cells (probably from Sigma Aldrich). In their protocols, it gives a culture medium of DMEM (without L-Glutamine) + 10-15% FBS + 2mM Glutamine. As seen here:

    However, my lab uses DMEM from Gibco which already contains L-Glutamine. As seen in this link:

    Looking these two media types, I noticed that the Gibco one has lower gluocose levels as well as other differences. Can I use the Gibco DMEM instead of purchasing new DMEM and glucose separately? Will the difference in glucose levels need to be substituted?

  6. Bigga Lingam

    I have a hard time estimating confluence. Looking over others’ shoulders, these 30% and 70% seem one of the most sinister jokes in molbio.
    If you are saying that time between splits is the time it takes to go from 30% to 70%, that means you are splitting them at every doubling. Various Pubmed-found articles say that C2C12 cell divison occurs every 19 or even 12 hours.
    When do you find time to change the medium if you have to split daily?
    I split 1:40, but if you are to go from 70% to 30%, how do you do it? 1:2?
    Do you count the cells?
    Sr. labmate claims pyruvate is poisonous to C2C12, but I find no published evidence. Is there any peer-reviewed literature on the ideal DMEM for C2C12?

    1. FD

      Hi Bigga,

      I agree that it is hard to estimate the confluency of the cells and it is very much depending on the person. Therefore I decided to always count the cells when passing them. Only then you seed the same amount every time it is reproducible. What is important with culturing C2C12 cells is that they don’t grow confluent (unless you want to differentiate them), so I use fixed cell amounts to pass. When I culture them every other day I use 1.5e6 cells in a T175 flask, for 3 days I use 4e5 cells in a T175 flask.
      The medium I use doesn’t contain pyruvate, I use DMEM 61965 of Life Technologies with high glucose.

    1. Absolutely. The reason for switching to HS instead of FBS is mostly related to cost (HS is cheaper, and you’ll burn through a lot of it changing your diff media daily).


  7. Ela

    Dear all,
    I would like to ask if it is possible to use differenciated myotubes for oxygraph (Oroboros) experiments? For that first you have to tripsinize and incubate them for 30min in Respiration medium (MIRO5) together with digitonin in 4’C. Do you think that after this procedure cells will be still alive ?

  8. Anmol

    Thank you for valuable information provided here. I have been working with C2C12 cells for last 3 months. I have transfected C2C12 cells with siRNAs using RNAi max (Invitrogen) and after 48 hours of transfect in growth medium( DMEM+10% FBS+Antibiotics,GM), I starts differentiating by adding differentiating medium( DMEM+2% horse serum+Antibiotics,DM) and trouble starts here as cells starts to peel off and fold back. I tried Poly-L-ornithine to coat wells before seeding the cells but it does not help at all ! I am changing DM every 24 hours as recommended in protocol. I am looking forward to get some advice from you to sort out this problem. Thank you very much in advance!

    1. FD

      Hi Anmol,
      I’m not sure if you were able to find a solution yet, since it has been a while since you posted your problem.
      But maybe I can do some suggestions:
      Try optimising the number of cells you plate, it might be that with a slightly lower amount of cells when starting the differentiation it will go better.
      For coating I have used Matrigel, this works very well. Dilute the Matrigel 50x in growth medium and incubate for 1 hr in the incubator prior to cell seeding. Rinse the Matrigel with PBS or medium.

      1. Maria Sara Magarò

        Dear FD,
        I’m Maria Sara Magarò, I’m an Italian PhD student working on C2C12 and I would like to differentiate them and study the maturation process via Immunofluorescence. I’ve already tried gelatin-coated glass coverslips, but myotubes detached on day 3 of differentiation.
        At which concentration did you use Matrigel for coating?
        Thank you in advance!

      2. Francina

        Hi Maria,
        I used Matrigel 50 times diluted in medium without additions. You should keep it on ice during preparation to keep it fluid. I now don’t use Matrigel anymore, but just culture the cells on the well plates without coating. This works very well for me right now.
        Good luck!

  9. hello every one,

    I started working with c2c12 one and half a year ago, initially i am satisfied with my cells and results but recently i am getting lot of problems with black dots in the culture and improper differentiation and my RNA yield is only .4-.6 ug/ul (initially it was 3-5ug/ul). so i want to know what could be the reason for this i am eagerly waiting to solve this problem i have tried many ways but no use. can any one help to get rid of this problem.
    this is my media

    1. A. de Boer

      Hi, regarding the black dots I experience the same. My culture has a lot of them, but the medium does not change to yellow. So others say it cannot be infected. But I am not convinced.

      1. Linda

        I also get the same thing, black particles in culture, what could it be? Do you observe this with higher passage numbers?

  10. Bigga Lingam

    I wish we had a better discussion forum than this. Thank you all for answers.
    Here are a few ideas that I wanted to submit to discussion:
    Did anybody try citarabine after differentiation? (mentioned in a few papers) Do the tube live longer or less without the dividing cells?
    Did anybody dare feeding tubes with serum-free medium? In fact, what do you people give them after differentiation, and how long do they cells survive in your choice of post-differentiation medium?
    I wnat to run some experiments on tubes, and I find it pure mess to tolerate serum in the environments and dividing cells in the dish.

    1. Bigga Lingam

      First to answer should be me.
      Never used citarabine, hence the question.
      I used 10% FBS and 2% HS for the post-differentation stage, and the cells did not seem to live past day 10 post-differentiation, regardless of approach.

    2. FD

      You are right about the discussions on this forum!
      When I work with differentiated tubes I incubate them for 4 hr in serum free medium and also my eperiment is without serum and takes 1 hr.
      I have never used citarabine.
      Good luck!

  11. FD

    Hello everyone,
    Does anyone have experience with differentiating C2C12 cells with special serum-free medium, called Aim-V serum-free medium (from Life Technologies)?
    And I would like to know your experiences with plate coating before differentiation.
    I have a very nice paper in which several comparisons are made in serum and coating, from M. Lawson, Cells Tissues Organs 2000, 167, 130-137. Very interesting, although already very old..
    I’m interested in your opinion!

  12. KM

    Hi, Can someone tell me the best primary and secondary antibodies to use with the C2C12 line as I am new to the model and my budget is limited. I need an antibody for MHC, Myogenin and MYOD along with a secondary for use in Western Blot. Can you please include the manufacturer ref numbers as well please as this will help me greatly.

    1. Hey there KM,

      I find that the best pan-myosin antibody (for both IF and WB) is the MF20 antibody from DSHB. This will also help with your budget limitations. As for MyoD and MyoG, your best bet might be to order from SCBT – the MyoD(C-20) and the MyoG(F5D) antibodies.

      I should really put together a page with antibodies, dilutions etc. for WB and IF. I’ll try and get around to this.


      1. KM

        Hi James, I will look into them. Can you also help me with another teething issue I am having with this cell line. Basically the cells (currently P7) are growing beautifully in t75 flasks, when left to differentiate in flasks and supplemented with DM they differentiate perfectly, but when I seed cells in to 6 well plates ( 100 000 cells per plate) and grow for a few days in GM ( they generally look fine at this stage) and then replace with DM they start to die off in huge numbers. On looking down the microscope, it appears they start dying of from the sides of the plates, forming what looks like little cirlces in the monolayer, before then peeling off in huge numbers leaving completely cell free bottom apart from areas around the centre, myotube formation never seems to get going and most of the cells are just clumped and floating around by that stage anyways. Cells continue to grow in flasks and when testing the differentiatingfcapabilities in a spare flask, can still do so perfectly. What am I doing wrong?

  13. ak

    Could anyone suggest a protocol for C2C12 differentiation on Glass coverslips? My experiments are for a total of 6 days long, with differentiation initiated at 48 hrs. I can get the cells to differentiate beautifully on 6-well plastic plates, but not on glass coverslips. Anyone else facing the same problem?
    I have tried these strategies so far-
    1) coating with poly-l-lysine: Doesn’t work
    2) coating with poly-l-lysine and laminin: doesn’t work. The cells start to come off the surface of the coverslips in clumps on Day 5. Probably contraction?
    3) collagen-coated matek dishes: cells differentiate, but like someone else mentioned, the cells come off the surface. And this is very noticeable when I perform knockdowns or put them in other stressful conditions.
    Has anyone used matrigel-coated glass coverslips for confocal microscopy and with siRNA knockdowns?


    1. RRW

      Have had similar problems, and never got a good IFA done using glass regardless of the treatment.
      The ONLY thing that really worked for me for IFA were ibidi ibiTreated u-slides (e.g. 8-well plates). This way they don’t need to be moved when ready for staining and you don’t need to worry about treating the glass coverslip, and they really prefer the plastic over glass it seems. And there are various treatment options.

  14. Ryan

    Just wanted to show my appreciation James, this level of methods is always so difficult to work out and saves loads of time. Cheers

  15. RRW

    Hi there, thanks for all this info everyone. I am thinking about moving into a human skeletal muscle (myoblast) cell line but don’t know where to start.

    Specifically I’d like to know:

    a) which cell line to use (there are some commercially available from GIBCO for example)
    b) whether or not they are similar to C2C12 cells in terms on media requirements, longevity and passage number with retained differentiation capacity
    c) whether anyone knows if they can be transduced using lentivirus (as C2C12 cells can)

    Thank you.

    FYI: I am also posting my lentivirus transduction protocol for C2C12 cells (below)

    Lentivirus production:
    HEK 293T cells have been modified to express the simian virus 40 (SV40) large T
    antigen. This allows the replication of episomal (non-integrated) plasmids that
    contain the SV40 origin of replication (oriC). HEK 293T cells were seeded into 15 cm petri
    dishes 24 hours before transfection. Fresh HEK 293T growth media was added. A
    mixture of the mammalian (lentiviral) expression vector and the
    lentiviral accessory plasmids was prepared, (accessory plasmids = pCMV-VSVG, pCMV-Gag/Pol, pCMV-TAT and pCMV-REV) using the relative amounts listed in Table 2.1. (Refer to Trans-iT manual). Cells were transfected with the plasmid mixture using TransIT-293 Transfection Reagent (Mirus Bio) according to the manufacturer’s instructions, using Opti-MEM I reduced serum media (Gibco). Growth media (supernatant containing lentivirus particles) was
    collected every 24 hours for 72 hours and replaced with fresh growth media. Supernatants containing virus were pooled and sterile-filtered (0.45 μm pore size) before concentration of lentivirus by centrifugation at 20,000xg for 90 minutes. Lentivirus was resuspended in fresh C2C12 growth media (or differentiation media) and used directly for transduction.

    Lentivirus transduction of C2C12 myotubes:
    For C2C12 myotubes, myoblast cell lines were counted, seeded into a 6-well plate
    and differentiated. Three days after the addition of differentiation media, cells were
    transduced. Lentivirus in differentiation media (2 ml per well) was supplemented with Polybrene (Sigma-Aldrich, 8 μg/ml) and added to each well. Cells were centrifuged with the lentivirus for
    90 minutes at 2000xg for “spinfection”. Fresh differentiation media was then added to the cells 6 hours post-transduction.

    Lentivirus transduction of C2C12 myoblasts and generation of stable lines (inducible expression system):
    For C2C12 myoblasts and generation of stable cell lines, cells were counted and
    seeded into a 6-well plate 24 hours before transduction to ensure 70% confluency for
    transduction. The control vector (pLVX Tet On) lentivirus and the expression vector
    (pLVX Tight Puro) lentivirus (in growth media) were added to the cells (with 8 μg/ml Polybrene) at the same time for simultaneous transduction (1 ml each per well). Transductions were
    carried out as described above and 48 hours later, geneticin (G418, 500 μg/ml) and
    puromycin (4 μg/ml) were added to the growth media for selection. Control cells were
    completely dead after 5 days of drug selection. Cells were maintained in growth
    media supplemented with 250 μg/ml G418 and 4 μg/ml puromycin. Stable C2C12
    cell line protein (of interest) expression was induced using 2 μg/ml doxycycline.

    1. Jin

      I’ve tried the spinfection protocol for C2C12 myotubes, but the myotubes seem to lift off the coverslips after the centrifugation step. Has anyone tried spinfection at a lower g for myotubes and got decent transduction?

      1. RRW

        I often found the same problem with myotubes, and instead now I use a doxycycline inducible vector system. I transduce at the myoblast stage, drug-select successfully transduced cells, then differentiate into myotubes, then switch on expression on the transgene.

  16. wenbiao shi

    Hi, thanks for all this information everyone. But can someone tell me the differences between developing or growing C2C12 cells and differentiated ones? Why do we usually use differentiated C2C12 cells instead of growing ones when we want to allow them to be exposed to the experimental medium? THANKS in advance~.

  17. vvericly

    Hi everyone, it’s such a rare find to have a C1C12 forum. I am a new phd student and I have question (many questions actually). What happens if one were to replate a confluent plate of C2C12 myoblast, containing a few myotubes by accident? How does it affect my experiments on myotubes?

    1. Hi Vvericly, if you replate confluent myoblasts that contain myotubes you’ll find that your replated cells will divide very slowly (a sub population may continue to divide as normal, but most will exit the cell cycle). It is possible to save the colony but it is a pain, a better option is to just start a new aliquot and make sure the myoblasts remain between 20-60% confluent.

      1. pisi este cerce

        I tried to detach and re-seed myotubes, and failed. The tubes just floated, the only time when I tried, and presumably died. Are you sure they can re-attach?

      2. Hi Pisi,
        You cannot re-seed myotubes (as you discovered). What I was suggesting was isolation and re-seeding of the reserve cell population if Vvericly needed to replenish her cell line.


      3. Juanita

        Protocol to isolate myotubes from myoblasts:

        C2C12 myotubes were isolated and plated at low density after trypsinization and sieving through a 100-um mesh. Myotubes were retained on this sieve, whereas the mononucleated cells passed through the 100-um mesh. Myotubes were washed off the sieve and plated at either 7–14 myotubes per mm2 or about 2 myotubes per mm2 onto 35-mm plates precoated with 0.75% gelatin.

        I coated my plate with 5ug/mL fibronectin in a .02% gelatin solution and it worked great. Myotubes stay healthy and even contract!

  18. Simona

    Hi everybody! I have just started to work with C2C12 and I have a question about their differentiation. I am searching on internet to understand how my cells should look like (after differentiation), since nobody in lab can help me about this. I see everywhere these great pictures of plates with basically only myotubes and very few indifferentiated cells (like this: Is this the normal rate of differentiation they should have? I mean…are they taking pictures of areas particularly nice to see or all the plate should be like this? Can someone send me a picture of a good differentiation experiment performed by himself?
    Thank you very much 🙂

    1. Leo Strehaya

      IMHO, timing of seeding and shifting is paramount, as is the number of pasages the cells have seen. There is a paper showing that higher density of cells at seeding was beneficial. Still, it felt like luck when I got extremely long tubes.

    2. Zahra

      Hey Simona,

      I got pictures of my 5-day differentiation I could send you for the ONE time that it actually worked properly for me. I’m not sure how to upload it here though…

    3. Juanita

      To increase your differentiation. Use DMEM high glucose with no sodium pyruvate, 2% heat inactivated horse serum, and L-glutamine. DON’T USE GLUTAMAX!!!!! It slows down the differentiation.

  19. Andreas

    I have a problem with C2C12 in 24-well-plates. The cells do not proliferate or differentiate with the the same rate. At first it seems that the inside wells work better. But after a few days many cells died in all wells. I work with DMEM with 10% FBS or 2% HS from Biochrom (1ml per well). Do you have an idea for this problem?

    1. Andreas,
      Can you include a bit more information?
      – what is the catalogue number of your DMEM (does it contain Na-Pyr? Glutamine or glutaMAX?)
      – 10% FBS in proliferating media and 2% HS in diff media?
      – how often are you changing the media?
      – are you maintaining the cells in 5% or 10% CO2?
      – what passage number are you up to?
      – how confluent are the cells when you start differentiation?

      1. Andreas

        Hi James,

        thanks for reply. The catalogue number of DMEM is FG 0435, the DMEM is without Na-Pyr and with stable Glutamine. Of course, 10% FBS is for proliferation und 2% HS for differentiation. I change the media after 2 days and I inccubate the cell in 5% CO2. The passage number is up to nine.
        I start the differentiation if cells are 70-80% confluent.


      1. I am using Glutamax and have been always using it without problems but the few last times I have been noticing lots of dying cells and very slow growth Rate , Why do you prefer using fresh L Glutamine instead ?

  20. Simona

    Hi everyone,
    I have question about C2C12. After differentiation I have in my dish a mixed population of Myoblasts and Myotubes. Is it possible to selectively isolate myotubes? And if it is possible, can you please tell me what is the protocol?
    I remember that I found something about this when I was collecting information about this cell line, but I am not able to find it anymore.
    Thank you! 🙂

  21. Allison

    I am a novice researcher and my first experiment is using the C2C12 line, specifically looking at the effects of myogenin. This article stated that myogenin can be tagged with immunofluorescence. I was wondering how exactly that was done? I am not sure of what will or won’t fit into my budget, so I need to know the protocol. I have a fluorescent microscope available to me.
    Thank you.

  22. Elliot


    Just wondering if anyone else has noticed this problem:

    I have nice differentiation of C2C12 cells into myotubes on 6-well plates, but seem to be having difficulty getting cells to differentiate on smaller surface areas than a 6 well plate (12 well, 24 well etc).

    I initiate differentiation once proliferating cells reach >95%ish confluence and differentiate for 72 hours. I begin seeing myotubes forming after about 48 hours. Any longer than 72 hours and my myotubes begin lifting off.

    In case you are curious:
    growth media – DMEM high glucose, no sodium pyruvate, 20% FBS,
    differentiation media – DMEM high glucose, no sodium pyruvate, 2% charcoal-stripped horse serum, 1uM insulin (charcoal stripped horse serum because I work with androgens and so I want to get rid of endogenous androgens within the serum)


      1. Elliot

        Hi Simona, I do not coat my plates. I use Corning brand plates and the cells seem to adhere to these plates without the need for coating

      1. Hi Juanita,

        There is no need to coat your plates before you seed cells on to them, that’s not to say you shouldn’t (and there are some very good reasons to coat your plates) just that it’s not essential.

    1. Alessandra

      I study Mineralocorticoid Receptor and I’m doing some exeperiment with C2C12 using Charcoal Stripped serum to avoid steroids presence in differentiation medium. The problem is that I have a small number of myotubes in plates differentiated with Charcoal serum compared to the others with 2%HS after 96h of differentiation.
      Did you notice the same thing? Do you have some advice to improve differentiation?

  23. C

    I start to work on C2C12 recently. And I found my c2c12 can form into myotube, but it takes more than 6 days!
    I initiate differentiation when cells reach more than 95% confluence. I can observe small myotubes around d6.
    Do you think the cells can still be used? Or you suggest to start using younger passage one? Or do you know any reason of this situation?


      1. ZAhra


        I’m having the exact same problem.

        I do difffertiate them in a stadndard way (DMEM+5%HS+1%p/S). I used passage cells from 9 up to 20 and it did not work. So I used a thawed passage 13 it worked But once I splitted again it did not work!!

        So I thought maybe 13 is the magic number. So I used another passage 13…and again it did not work. It does not differentiate. The few that do are short and fat-looking myotubes, quite different from how they should look.

        I’m going crazy. I’ve been working on this for months and I have a data presentation in less than a month…and I have nothing to present.

        I would truly appreciate your help!!!

    1. Simona

      Hi! I don´t know what passage number are you using, but my C2C12 can differentiate very well even at passage 27. I have never tried an higher passage. Obviously using them at a lower passage would be better, but I think that probably something else happened to your cells. Once it happened to me that I seeded them at a too low density and they started acting very strangely. In the end I had to throw them away and I thawed new ones. But I think you should specify better how you handle them to get a good answer.
      Bye 🙂

      1. Zahra

        Thank you so much for your response Simona!

        I basically let my cells grow to about 90-100% confluency, then I split them. But when I seed them, I put a minimal amount of cells so they reach confluency later so I would not have to split as frequently. Maybe the initial low confluency combined with prolonged time of growth is not good…

        But Let’s assume that there is something wrong with my cells. If I use a later passage from the same linage of cells that I had split (so I started off with a P9 and now I am at P16)…are all my cells of the later generation messed up assuming that “something happened” to them at a earlier passage? I’m not sure If I’m making myself clear on this…

        ‘Cause you mentioned that your cells began to act strangely and you thawed a new passage. So after you noticed something “weird” , that new passage was from another lineage of cells, correct?

        Also, can you describe what went strangely with your cells? Maybe the same has happened to mine. 😦

        Thanks again for your help. Means a lot!!!!!!


      1. Keeping passage numbers low is important (especially for you frozen stocks (always try to keep a few aliquots frozen from your original stock). Their should be no problem with using cells <passage 20. After passage 20, you just need to keep an eye on the rates of proliferation and differentiation (both will decrease over time). For primary cells, absolutely, passage 6 is the maximum you'll probably be able to get to before the cells senesce

      2. Part of my reply got cut off.
        -There should be no problem culturing cells out to passage 25-30 (provided your cells have come directly from ATCC and not a collaborator).

  24. julia

    Does anyone have a recommended freezing mixture for c2c12 cells. I am planning to freeze them undifferentiated. If you do freeze thaw does anyone have an estimate of % cell viability following thaw.
    best wishes

    1. Zahra

      Hey Julia,

      The post-doc in my lab suggests freezing about 500 microlitres to 1000 microlitres. Take 1% of the total volume as the amount of DMS-O to add. Don’t add too much since it is toxic. I know others add 10% of volume in DMS-O.

      Then snap freeze it (I usually do so in liquid Nitrogen). Then allow it to freeze overnight in -80C to allow a gradual freeze. The next day store in -120 liquid nitrogen.

      I don’t know the percent viability. But when you thaw it, allow the cells to grow in a small flask until they are confluent enough to split. Starting off with a small flasks allows the cells to reach confluence sooner and the close proximity of the cells facilitates faster growth.

      Hope that helped.


    1. Zahra

      No problem! But I forgot to mention that the very next day after you plated, you have to change the medium to get rid of the toxic DMS-O. The protocal I mentioned is in regards to cells diluted in medium (amount depends on how concentrated you want it).

      There’s different ways to freeze, of course. You can isolate the cells from the medium by centrifuging until you get a pellet. You can mix the pellet with 10% of volume in DMS-O and FBS (not sure how much, didn’t ask) then proceed to freeze as mentioned above.

      But I’ve never tried it this way. The other post-doc in our lab uses this method. I go with the first method: the cells diluted with my medium since it works well 🙂

  25. Hello Everybody,
    we are restarting the lab after it moved, an advice when it comes to thawing the Cells to split and replate, to have more cells, we currently have just one tube so we are afraid of losing all what we have. Any advice or particular protocol to follow for thawing the cells !

    Thanks a million

    1. Hi Nadine,

      Not sure if you have gone ahead and tried already, but in case you haven’t….
      I’ll assume your cells are C2C12 cells and are stored in a DMSO based recovery media, lets also assume you have an aliquot of ~200k cells that were frozen at a low passage number. Assuming all of this, the best thing to do is to plate your cells in growth media on a 10cm plate and leave overnight. Change with fresh growth media the next day and allow the cells to reach 40-50% confluency (or 3 days in culture, whichever comes first). Then split the cells in to 4-6 10cm plates and allow them to also reach 40-50% confluency (or 3 days in culture). At this point you should be able to freeze ~30-50 aliquots of 200-300k cells.


  26. shinyee

    Hi all,
    My project is about glucose metabolism and I would like to use C2C12 for my experiments. However I am not familiar in handling this cell line thus I have some burning questions and hope for advice.
    Does it matters whether the cells are differentiated or not for studying glucose metabolism? Also, is it possible to perform transient transfection or knockdown using sirna in differentiated C2C12 cells?
    Many thanks!

    1. Hi Shinyee,

      In answer to your first question, yes, glucose metabolism will be different in proliferating myoblasts versus differentiated myotubes. Which one you use will depend on the question you want to ask (and the technique you want to use). I will update the methods section soon with a detailed description of using proliferating C2C12 cells in the Seahorse XF24 bioanalyzer.

      For the second question, you can do siRNA mediated knockdown in differentiated C2C12 cells, it just wont be as effective as if you were to do knockdown in proliferating cells. A better option might be to generate a stable cell line using an inducible plasmid.


  27. Adam

    Hi I am doing a lab report on the proliferation and differentiation of muscle cells using DMEM and I am having difficulty research what it is exactly in the solution that cause the cells to proliferate or differentiate. So if anyone could help me I would really appreciate it 🙂 ! I know it must involve the difference of fetal bovine serum vs adult horse serum but I just can’t seem to wrap my head around it..

    1. Toomas

      You’re in the right area but not quite there. I think that you could use FBS or horse serum to differentiate, so that isn’t the difference you are looking for. There’s something else about the media that is responsible.

    2. Hi Adam,

      Toomas is pointing you in the right direction, and because it is a lab report I don’t want to just come out and give you the answer. I’d recommend going back and reading some of the original papers by Helen Blau (Blau et al 1983) and Yaffe and Saxel (1977) who first isolated the C2C12 and C2 lines respectively.

      Also, HS is used instead of FBS in differentiation media mainly because it is significantly cheaper (and it doesn’t compromise differentiation). Look back at my protocol above, what is different between the growth media and the differentiation media?


  28. RRW

    Hi all, can anyone help me with a protocol for inducing contraction in differentiated C2C12s myotubes. I sometimes see spontaneous contraction but I would like to induce more contractions and compare cell lines. I can see lots of electrical pulse stimulation protocols using various different equipment types, but what I’d like is to be able to induce contractions chemically, with calcium and ATP perhaps, if this is at all possible??
    Thanks very much!

    1. Jin

      Hi RRW,

      I’m looking for a similar protocol as well, would like to check if you have any updates on this? Was reading up as well, and seems like caffeine might be a plausible alternative, as caffeine appears to induce transient cytosolic Ca2+ via inhibtion of RyR (Reference: Store-operated calcium entry in differentiated C2C12 skeletal muscle cells).

  29. Chu

    Hi James, interesting blog and I have learned a great deal from your and contributors’ experiences. Thank you!

    1) I like to create gene KO C2C12 by using CRISPR. For other cell lines we use, the current protocol is to ligate gRNA into Cas9 GFP construct ( addgene), conduct transient transfection, and then do single cell sorting to 96 well plate. Then we will analyze the property of cell clones ( if there is any) by PCR , IF and WB. For C2C12, I think we will get some problems using this protocol; because if we allow C2C12 to grow into single clone after sorting, they will start to form myotube. I would love to get any inputs/suggestions/comments on how to overcome this problem before we do something stupid.

    2) do the myotubes differentiated from C2C12 express dystrophin?

    Thanks a lot!


    Chu, YS

    1. Hi Chu,

      Glad you’ve found the blog useful, I hope to add some updates soon. In answer to your questions:
      1) No, the protocol you have described should work perfectly (provided the gene you have KO’d is not essential for proliferation). Isolation of single clones is difficult, but certainly possible. One problem you will run in to is that only a small population of C2C12 cells will form colonies. Many of your single cells will simply senesce and apoptose.
      2) I believe differentiated C2C12 cells express dystrophin, but I don’t know whether it is limited to a specific isoform, hopefully someone else can provide a detailed answer to this?


    2. judith cossins

      Hi Chu,
      We are doing this type of work and have run into a few problems that we didn’t foresee. One problem is that C2C12 cells are polyploid. When you make a knock out cell line using CRISPR/Cas9, you cut the DNA and rely on NHEJ to repair the DNA, and you are looking for the repair on all alleles to be out of frame. But if you have 5 or 6 alleles, each has to be repaired out of frame. It’s a big ask and we find a lot of our gene edited clones have a deletion or insertion which is in frame on at least one allele. It also makes it really difficult to analyse simply by PCR and sequencing the PCR products. Often we have to clone the PCR products, grow colonies and have lots of colonies sequenced. You have to screen a lot of clones. Another problem, maybe a consequence of the polyploid nature, is that clones can have different differentiation properties. Some don’t fuse. Some fuse in medium with 2% FCS, whereas others need a bit of horse serum as well. Even if you clone a clone you still end up with this variation in differentiation ability. Because of this we also have several control CRISPR/Cas9 clones which have been transfected with an empty pX330/335 vector and cloned. We then analyse several and get an average – usually looking at ACHR clustering. We don’t tend to see the cells differentiating in the 96 well plates when we are cloning them, so I think that will be the least of your problems!

      I’m afraid I don’t know about dystrophin.

      Best wishes and good luck!

  30. HY Chan

    HI James, Thanks for your sharing. I am an engineer and new to C2C12. My problem is that I can sub-culture and differentiate the C2C12 cells. However, I cannot see any spontaneous reaction of the myotubes. I don’t know if it is a MUST or just by luck.

    I followed your protocol and I can see some tubes at the end. However, I found that my cells is different from others that no matter how, I can see some cells that haven’t yet differentiate in the flash. I cannot make the whole flash differentiate into tubes. Is that the problem of why I cannot see spontaneous reaction?
    for GM, I used DMEM with 10%FBS, 1% PSN.
    for differentiate one, I used DMEM with 10% Horse serum and 1% of PSN
    Also, it took few days for the differentiate to happen.

    Thank you.

    1. Hi HY,

      I assume you mean that you cannot see any spontaneous contraction of your differentiated myotubes? Here are a few points to consider:
      1) You need to lower the amount of serum in your differentiation media, 2% horse serum is standard and works well.
      2) Regardless of how long you incubate the cells in differentiation media a small population will not differentiate (these have been referred to as the reserve population).
      3) Finally, spontaneous contraction of myotubes will only occur during the late stages of differentiation (often past 5 days post-differentiation).

      1. HY Chan

        Hi James,
        Really thanks for your prompt and kind response.

        Yes. I cannot see any spontaneous contraction.

        1. I used 10% horse serum as stated in ATCC specification. But, I do read lots of people used 2% only. I will give a trial on that. Thanks for your advice. I also read people added ITS or Cytosine. Would you recommend?

        2. I see.

        3. 5 Days of post-differentiation. Is that mean 5 days after I switched to GM with 2% horse serum?

      2. If you really want to push the cells to differentiate rapidly, you can add ITS to your differentiation media (you just have to keep in mind, you are activating several hypertrophic pathways).
        Yes, I mean 5 days after you switch to differentiation media.

      3. HY Chan

        Hi James,

        I have tried different methods for differentiation. However, I have no luck in seeing any spontaneous contraction. I have read some papers saying if I cultures the cells at 100% confluent. It will deplete the myoblastic population. I remembered I harvested my cell at 100% confluent when I first got the cell from ATCC. Will that be the reason why I cannot get any spontaneous contraction from this cell line?Thanks


    2. HY Chan

      Dear James,

      I have tried your recommendation on using 2% Horse serum for differentiation. However, I still cannot see any spontaneous contraction. Also, I have tried using electrical pulse to simulate it but no luck before I see many electrolysis happened. Do you have any recommendation on how to check whether my myotubes are the “real” one? Thank you

      1. You’ll really need to push your differentiation (6+ days, change media daily). Remember that myotubes are not myofibers, staining with MF20 (a pan-myosin antibody from DSHB) will not show striations. Depending on what you want to do, C2C12 cells may not work for your desired study. You might be better off with isolating single muscle fibers.

      2. HY Chan

        Thanks. James. Let me try your recommendation. I didn’t change the media daily. Instead, I changed every other day. Will that make that much difference?
        What I want is actually use it as an bioactuator. It is not new. I am now following some paper work on using it as actuation. But, I have a difficulty in making it contract.

  31. Adam


    I am having issues with the cells proliferating in a 12 well plate. The cells are predominately settling in the center of each well, leaving a large area of the well empty by the time the cells in the center of the well reach 70% confluent.

    I am using DMEM and 20% FBS.

    Any tips to prevent this or any advice would be greatly appreciated.


  32. Ankita

    hi!! Very recently I have started working with c2c12 cells and I want to differentiate them treat them with adenovirus and then induce muscle degeneration using dexamethasone. I am using 2% horse serum but without insulin transferrin. Cells grow fine till I change the media to horse serum media. My cells differentiate but very slow and after adenovirus infection i see a change in the morphology of cells. Along with that i am using a drug called decadron for dexamethasone. Is it fine to use them or should I order for molecular grade dexamethasone n can you plz suggest me how to dissolve the decadron and what should be the concentration. My cells were of passage7-8.

  33. Roni


    What will be a recommended transfection reagent and protocol for transfecting non-differentiated C2C12 in 2D using a non-viral delivery method? and encapsulated C2C12 in hydrogels?

    Thanks a lot

  34. Ting

    Hi James,
    I have wasted 2 month in transfection of differentiated C2C12. The cell looks good before add transfection reagent(use siRNA and lipo2000)with completed differentiation,and looks also
    good after overnight transfection. The problem is when I changed the transfection reagent to DM, the cell starts to peel off and fold back.
    I tried many ways of change medium. Complete remove the reagent and add warm DM, do not remove the reagent and add warm DM directly, remove half the mix of reagent and DM then add new warm DM. However, it didn’t help.
    Occasionally,the myotube survived after 48h transfection,the knockdown efficiency was near 80%。

    Condition: Seed the cell in GM for two days, then change the medium to DM, change the DM every for four Days, the myotube looks good.

    Thanks a lot

  35. Mehtap

    Hi James,
    Thanks for this great website!
    I have the following question: How are you making cell lysate of differentiated C2C12 cells?
    My goal was to separate the differentiated myotubes from the remaining mono-nucleated cells by mild trypsinization and to make cell lysate of both fractions, which I want to use in Western blot analysis.
    The volume of the cell pellet of the fraction enriched in myotubes (detaches faster than the mono-nucleated cells), and the one of the remaining cells enriched for mono-nucleated cells looked about similar.
    However, when making cell lysate of both fractions (by resuspening the cells in RIPA buffer with protease inhibitor cocktail, and leaving them on ice for 60 min and then centrifuging to get rid of cell depris) I got a yield of about 1000 microgram for the mono-nucleated fraction (starting from a 10 cm cell culture dish) but only about 150 micrograms for the myotube enriched fraction.
    Is there another protocol of cell lysate preparation that works better for the myotubes? Or are they maybe too fragile for centrifugation at 200g (when I collected cells in the beginning)?
    I would be happy if anybody could help me with that!

    Thanks in advance!

  36. Olga

    HI there,
    Can anyone help me with a suitable marker to check up the C2C12 differentiation in qPCR? I got to compare c2c12 with three different protein KD. At what day C2C12 are considered fully differentiated? are 2-3 days enough? if I do longer like say 5 days, would it be inaccurate? I am thinking of MyoG and MyoD1 markers, is their expression kinda linear through the differentiation? I mean does it keep going up the later the stage is or it jumps up and down over the time?
    thank you!

    1. Hi Olga,
      There is no day at which C2C12 cells are considered “fully differentiated” as they only ever reach the myotube stage. Depending on your culture conditions, myogenin will appear at ~12 hours after the induction of differentiation, and reach a peak at ~2-3 days. Embryonic myosin (Myh3) will appear around day 2-3 and keep increasing. Using these two markers, you can investigate early/late differentiation.

  37. Carolina

    Hi James, What do you know about C2C12 suface integrins which participate in adhesion to SFB proteins coated in plates in cell adhesion assays. Do you have any article to recommend me? Thanks!

    1. Unfortunately, once C2C12 cells reach confluence, they will rapidly exit the cell cycle and differentiate. If you try to split these cells and keep growing them, you will rapidly see a decline in the rate of proliferation. More importantly, you’ll have a mixed population of proliferating/differentiating cells which will influence your results.
      Always avoid letting your cells get much above 60-70% confluent.


  38. I also split cells 1 P10 into 8 P10 plates –> they proliferated into islands ! is that normal ! or is it because i seeded not much cells in each plate ?

    1. Saidnm,

      If you seed the cells at a density of less than 10% then you will see the formation of individual colonies. Try seeding at a slightly high density, hopefully that will solve your problem.


      1. thank you James , yes may be they were too little ,
        i thawed another vial of the same batch coming from the same ATCC vial, it grows much better, but I see lots of senescent fried-egg like looking cells and somehow the C2C12 looks a bit weird and elongated . is that a problem of the cells in the first ATCC vial itself ! or is it something in the media or culturing and splitting that triggers this !

        best regards

  39. Mehtap

    Can anybody recommend an antibody (For Western and IF) I can use to mark the undifferentiated state in C2C12 cells?

    I have anti Myf5 antibody (sc-302 of SC), it used to work but with weak signal and now it doesn’t work anymore.
    I don’t know if I should get the same one again or if there is something better I could use..

    Thanks in advance…

    1. Mehtap,

      Pax7 will work (although this won’t mark 100% of undifferentiated cells, as proliferating C2C12 cells are a mixed population). Why don’t you use the absence of Myogenin to mark the undifferentiated state?


  40. Rashmi

    Dear All,

    I am facing the problem of myotube layer coming off during washing step of my assay. I wasn’t facing this problem earlier, until few months back. I grow them in 96 wells plate at seeding density of 20k. We changed FBS lot and I shifted to DMEM with GLUTAMAX. IsDMEM-GLUTAMAX responsible for the observed effect (maybe causing higher proliferation even in the differentiated state)? I reduced % FBS from 2 to 0.5 and there was some relief, but the result is not consistent. For example, half of the wells show this behavior after differentiation. I observe that layer peels off from the edge and forms clump of the cells. Your suggestions would be valuable.


    1. Rashmi,
      Can you provide more detail about your conditions? How long after seeding do you switch to diff media? How long before the cells start to peel off? How often are you changing the diff media? Is there Na-Pyr in your diff media? Are you using DMEM with 25mM glucose?


    2. Dan

      Hi Rashmi,
      I have the same problem. Upon induction of differentiation the cells peal-off from the edge and finally the monolayer of cells floats into the well. Did you solve the problem? Is it due to glutamax?


      1. Rashmi


        I can say I have kind of solved the problem. For information, I am using DMEM-GLUTAMAX (4.5gm Glucose/ standard medium) with 10% FBS for seeding. I realised that the problem started with new batch of FBS. Probably too rich in nutrients ! So I reduced the % of FBS in differentiating medium from 2 to 0.5 %. There was some relief after this. But then I reduced initial cell density from 20k to 10k, and now cells are doing fine. They are not coming off the plate while doing assays. GLUTAMAX or Glutamine may not make a difference. FBS will do. You can also try heat-inactivation of FBS for differentiation (just as an option), though I didn’t need to do that. So you can check whether, you have changed something, some media component, might be the clue. Hope this helps !


  41. Rashmi

    Dear All,

    I have another query regarding Palmitate treatment to C2C12 myotubes. If anybody using BSA-conjugated-palmitate to treat the cells, I would like to know do you boil the final 5mM stock of 10%BSA-Palmitate before giving the treatment and why it is required ? I usually boil it at 55c for 15 minutes. Last time I tried without boiling the stock and observed lot of cell death even in BSA-treated control wells. I don’t understand why this would happen. Why BSA would lead to cell death ? I can give detail protocol if required for the discussion.


  42. Hi guys 🙂

    I’m having a bit of C2C12 cell trouble too if anyone can help!

    My frozen p2 alliquot of cells when plated grew far faster than I thought (10%FBS, PSA, high glucose DMEM). They definitely became too confluent, however when split they seemed to still proliferate well.

    However when seeded in 24 well plates in a matrigel-collagen hyrdogel no fibre tension would form between our 2 anchor points (when I did it in my other lab it formed in 1 day!). One well looked dead and the other the cells began to aggregate however they didn’t form tension.

    Do you think these is because of over confluence previously? Has anyone else experienced the same trouble?

    I was going to PCR and check for differentiation markers to see if this has happened, if so and some have differentiated, should fibres still form?

    thanks so much,


  43. Dan

    I tried to use PLKO-Tet-on system to construct stable shRNA knockdown in C2C12 cells. Interestingly, I have alreday obtained perfect target gene knockodwn single clone in Hela cells. 90% Hela cells infected with lentivirus survive.

    But, when I try the same protocol in C2C12 cells. After puromycin 2ug/ml treatment for 2 days. Cells died a lot and there is no difference between control and samples infected with lentivirus. I used lipo to do transfection and do spin infection of C2C12 cells at about 65% confluent. I also tried to 2 round infection it still did not work. After 18h of transfection, I change the medium into 30%FBS to produce virus. The virus I collected were not concentrated. I use 1 well of 6-well plate to produce virus and just add the supernarant from this 1 well to infect 1 well of C2C12 cells. I use polybrene at 8ug/ml and after about 24h of infection I change the medium with DMEM10%FBS with antibiotics. After another 24h of incubation I added puromycin.

    Could anyone who may give me some suggestions? Thanks so much!

    1. Xue Sun

      Hi Dan,
      I met same problem with you, I transfected C2C12 cells using Neon Transfection System and the transfection efficiency is around 80% .But when I add puromycin medium after 24 hours of transfection and for another 48 hours, cells died a lot and there is no difference between control and samples. Do you solve the problem now?

  44. Matias Monsalves

    Dear James, im having problems with mine C1C12 when i passed them to the cover glasses (passed on 70% confluence to the 6-well plates). After 3 days of differentiation they start to detached from the cover glass!!
    Hope you can help me with this issue!

  45. Dinesh


    I am working with C2C12 myoblasts and I use DMEM with 4.5 g/L glucose and 10% FBS as growth medium. I regularly serum starve my cells for studying insulin signalling pathway. In several instances, I have serum starved them overnight without any issues. However, in the past couple of weeks, my cells start dying (become circular/detach) when I serum starve them, in less than an hour. Same thing happens when I starve them in serum-free low glucose media (for studies needing low glucose). It happens whether or not sodium pyruvate is present. Does anyone know what might be wrong?


  46. Hanna

    I am having trouble to differentiate C2C12 cells.
    I have new passage (2-3), I plate them and split them when they are about 50-60% confluent. Proliferation medium is DMEM + 10% FBS. I add differentiatio medium when cells are more than 60% and medium contain 2% horse serum. My PI have opinion that cells should be passage in very low density (cells should not touch each other, because when it happen they can start differentiate). Also, he suggests that in 48 h cells should have already plenty of myotubes and there is something wrong with cell culture if they need 5 days to differentiate. So far, my C2C12 didn’t differentiate at that time. Around 48 hours they start making myotubes, and there is more of them at 72 hours but they are thin and usually no more than 2 nuclei. In my previous lab we used C2C12 and we culture cells in DM medium for 5 days. What is your opinion about that? Are there any “standards” for differentiation of cells? Is that mean that my cells are culture in wrong way if they don’t differentiate in 3 days?

    1. KK

      LIke if I read my own old issues 🙂

      Did you read through advises above?

      1. Horse serum -> much better if it’s heat inactiated
      2. pH – What’s the pH pf your medium? They differentiate better in 7.5%CO2 than 5% for me
      3. If they touch each other a bit, should not be a problem. Passage number doesn’t change much for me
      4. 48h hours I start seeing myotubes. They are small though
      5. Manuela suggested above not to use medium with pyruvate.
      6. You can try to add glutamine
      7. Or you can boost them with ITS supplement

      8. Still, there are differences between my previous and current lab.

      Good luck!

    2. Mehtap

      Hi Hanna,
      For me they differentiate best if they are more dense: I let them grow to 80 – 100% confluency and then put them in DMEM with 5 % Donor Horse serum. If they are less dense, they simply don’t have who to fuse with and the differentiation isn’t so good and also takes longer.

      Good luck…

      1. Hanna

        Hi Mehtap and KK,
        Thank you very much for your answers! I am using DMEM medium w/t sodium pyruvate and with glutamine, so I don’t think that this is a problem. I already added them inactivated horse serum and ITS. If that will not help will try to keep them more confluent for differentiation.

  47. Matias Monsalves

    James: Hope you can help me. Im having problems with the C2C12 when i seeds them on glass covers for microscopy. After the forth day of differentiation they start to come off the glass from one of the sides. I change the medium and wash them with PBS really slow and carefully but still they came off.
    Any idea why this might be?.

    Really appreciate your help

    1. Adam

      HI Matias,

      I had the same problem but have managed to rectify this by coating the glass chamber slides with poly-l-lysine. This seems to do the job nicely and stop the cells from lifting.

      Hope this helps.

  48. Nadine

    Hello everybody,

    I wanted to start knocking down certain nuclear candidate genes in C2C12 myotubes . and have never done any knockdown before, anyone has a protocol to follow for myotube knockdown of genes.
    also anyone tried knocking down in the P10 or P15 plates , or i have to use 6 well plates ?????
    this is very confusing
    you will be much appreciated.
    best regards

    1. Martin

      I used siRNA in myotubes to knockdown genes before. I used siGENOME siRNAs from Dharmacon with the Dharmafect1 transfection reagent. You can buy for example 5nmols of siGENOME smartpool for your gene of interest. Shortly, I differentiated C2C12s for 3 days in Differentiation medium (DMEM D5796 with 2% Horse Serum and 1% L-Glutamine) and added the siRNAs at 25 nM concentration using 4-6 µl of Dharmafect1 per 6-well. There’s protocols by the manufacturer for everything.
      I worked with 6-wells, but other sizes should be possible.

  49. Ashok Mandala

    I did siRNA transfection in differentiated C2C12 cells using RNAimax from invitrogen. It worked well for me. I did transfection in 6-well plate 60mm dish as well as in 96-well plate. In all the cases I got it perfectly without any problem

    1. Ahmed

      Hi Ashok,

      Thanks for sharing your experience, What was the amount of siRNA and volume of RNAimax that you used? I tried 100 pMol to 1nMol (using Lipofectamine2000) but with no success.

      Best regards

  50. Pablo Morales Campos

    Hi everyone! First, I want to say what a good idea of forum this is! C2C12 cells are so difficulte to work with, that all the help is welcome.
    Im working with tubes after 5 days of differentiation, and then treating them for another 48h. I have to stimulate my cells with BSA plus some fatty acids, and since a few months ago, when I use my DM suplemente with the BSA:FA complex, the tubes start to look ugly, leaving “holes” in the plate and start to detach…it´s like if they started to contract. Can someone give me some guidance? Im losing all the culture by reaching death end of my experiments (cells look so bad, I have to throw them out 😦 )
    Thank you so much!

  51. Phil N

    Hi all,
    Thanks for all of your comments, they have really helped me to get started with C2C12 cells. Using the info here I’ve got some cells to differentiate into myotubes and I am hoping to now determine the fusion index/myotube formation percentage compared to myoblasts. Has anyone tried to automate this with imaging software, or do people just count nuclei in/not in myotubes?

    Pablo: I haven’t experienced the dark holes you are talking about, I can leave my cells differentiated until at least 10 days, the most important thing is to keep refeeding the cells with the differentiation media, are you doing this?

  52. Hi
    Has anyone been successful in making any kinds of knockdowns in C2C12. I have tried multiple times ad failed. I am talking about transducing myoblasts and maintaining those cell lines.

    1. Toomas

      Just coming across your question so I’m sorry it may be too late now, but hopefully this can still help others! I had no success using siRNAs, but I have had a lot of success using an adenovirus with an shRNA. This has even worked in fully differentiated myotubes.

  53. Ashok Mandala


    Has anyone successful in detecting lipid accumulation in C2C12 cells by oil red o staining?
    If yes please share the protocol. I have been trying in differentiated C2C12 cells but was not successful.

  54. Hi,

    I do not know if you guys will be able to answer this. I have looked everywhere and cannot find if myotubes differentiated from myoblasts and mytotubes differentiated from adult skeletal muscle cells are the same. I have different results when I infect these 2 types of myotubes with AAV. But in my mind, they should be exactly the same, with the same markers. I am looking for a higher expression of Desmin, since this is the promoter I want to induce expression from.


  55. adlin

    hi everyone. i have prob in differentiating my c2c12 cell lately. they are rapidly form myotube on day 3 but after that they stop fused with each other. have anyone experience with this before? i even change my HS to another batch but the prob are stll there. so in the middle of the well there are alot of myoblast but at the side the myotube successfully fused with each other and form longer myotube. so the prob is in the middle. can anyone help me regarding this issue. thank you

    1. Francina

      Hi Adlin,
      Can it be that there are too many cells in the middle. Differentiation can be difficult with too less or too many cells. At the edges of the wells there are usually less cells, so this might explain the tube formation at the edges and not in the middle. To get a more equal distribution of cells, you can leave the plate for 20-30 min in the flow cabinet before putting it back into the incubator. This works very well for me with 96 wells plates. I hope this is of any help to you. Good luck!

  56. Linda

    When you say ” there are a number of important limitations that should be taken into account. ” Can you provide some examples or some references to that claim? thanks

  57. Panos

    First of all many thanks to James and all the scientists above for this great blog. I have started working with the c2c12 cells one month ago. I want to set up the AChR clustering assay. I use ATCC cells and my GM is ATCC DMEM 10 or 20% FBS (gibco 16000) non HI 1% PS. My DM is DMEM Gibco 12430 with 2% HS (Gibco or ATCC, HI) +- PS. I get some differentiation, but when I add human insulin (Sigma), differentiation is really great. I plate on treated plastic or autoclaved borosilicate coverslips +- poly L Lysine without major problems. I use R&D agrin and 1:1000 bungarotoxin alexa 594 or Cy5 from Invitrogen. I do not see staining and have tried pre and post fix with PFA (3% 10 min). Can anyone help? Is there something I am missing?

  58. Alia

    Hi, I am using C2C12 cells for MTM1 knockdown, you mentioned that the cells should be replated after it reaches >70% confluency. So it means I should infect them with lenti virus at 30-40% confluency since after infection we need to treat cells with antibiotics for 48 hours (changing media after every 24 hours)? Can you give me some advice on this?

    1. Hi Alia,

      It really depends on what your ultimate goal is. If you are trying to create a stable cell line, and you are using antibiotic selection, then the antibiotic should kill the majority of your cells across days 2-3. If you simply want to infect the cells and then differentiate them, then you could infect at 30-40% confluency and then change to differentiation media at 90% confluency.

      In general, you should split your cells regularly to keep them between 20-70% confluent. You should also avoid leaving C2C12 cells attached to the same plate for longer than 72hours.


  59. Mike

    Hi All,

    Thanks for the blogs.

    Has anybody tried to trypin and replate the C2C12 cells after differentiation? If so, should be the cell replated with differentiation medium or Growth medium? Thanks in advance!

    1. Mike,

      If you are trying to recover the “reserve cell” population (ie. the cells that have not undergone differentiation), then you should replate the cells in growth media.


      1. Mike

        Hi James,

        Thanks a lot for your kind reply. I want to know if the differentiated cell can still keep growth and stay in differentiation state when I trypsin and replate the differentiated cells in the growth media.



  60. Ellie Crompton

    Hello everyone,

    I have a similar question to Mike above. I would like to dissociate differentiated C2C12 cells and then re-seed these onto motor neurons to provide a co-culture system. I cannot dissociate my motor neurons as they are very fragile and will die, so I need a way to introduce the C2C12 cells. I do not think the C2C12 would differentiate if I seeded them onto motor neurons before differentiation.

    Any thoughts? Thank you!

  61. Patrick

    I see different versions of DMEM that contain either L-glucose or D-glucose, and even some that contain both. Given that D-glucose is the more common naturally occurring form of glucose, why do some versions of DMEM contain L-glucose. And should D-glucose always be used with c2c12 cells?
    Thanks for any thoughts.

  62. Alex

    Hello everyone,
    May I ask if anyone has ever looked at the percentage of MyoD immunopositive nuclei in proliferating C2C12 culture (i.e. in growth medium) using immunocytochemistry? Are they supposed to be 100% immunopositive or less? Thanks

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