Which is the best protocol to differentiate c2c12 cells. C2c12 cells were stimulated to differentiate into multinucleated, mhcexpressing myotubes in the 96well plate format by the addition of 2% horse serum in dmem. The cell cycle phases were analyzed by the flowjo software. Hi, ive been working for c2c12 s for quite some time now but only recently have started to have problems with differentiation with these cells. Cells were grown using dmem invitrogen, 10% fetal bovine serum, 0. Research paper dexamethasone treatment at the myoblast. C2c12 myoblastoma cell differentiation and proliferation is. I prefer change fresh medium everyday for 57 days to get the.
Creatine prevents the structural and functional damage to. The myogenic differentiation program is regulated primarily by the mrfs that all. Differentiation of the myoblasts was induced by serum starvation and. Characterization of fer1l6 in the mouse c2c12 cell line. Cells in a 6 well dish, plated and at 70% confluence. C2c12 differentiation was assessed as the extent of myotube formation, glucose metabolism, protein expression, sarcomere formation, and active tension generation.
After treatment, the cells were fixed, permeabilized and incubated with an antibody against the differentiation marker, myosin heavy chain. Damage to the myogenic differentiation of c2c12 cells by heat. Evaluation of serumfree differentiation conditions for c2c12. We observed the cell morphology by giemsa staining to. The differentiation state of these cultures was then tested both microscopically and biochemically. Reports of myostatin inhibiting c2c12 differentiation 5, 6, 22 are somewhat inaccurate because these studies demonstrate a delay in the process only after it has already been induced with confluencey and serum withdrawal. To induce myogenic differentiation of c2c12 cells, subconfluent cultures were switched from gm to differentiation medium dm consisting of dulbeccos modified eagles medium. Myostatin stimulates, not inihibits, c2c12 myoblast. Skeletal muscle growth and regeneration depend on the activation of satellite cells, which leads to myocyte proliferation, differentiation and fusion with existing muscle fibers. Developed for in vitro studies of myoblasts isolated from the complex interactions of in vivo conditions, c2c12 cells are useful in biomedical.
The chemokine sdf1 controls multiple steps of myogenesis. Sparcl1 promotes c2c12 cell differentiation via bmp7mediated. Interactions between skeletal muscle myoblasts and their. Owing to its ability for rapid differentiation that can be easily modulated by treatment with proteins, the c2c12 cell line has proven to be a reliable transfection host for. The c2c12 cell line is a subclone of myoblasts that were originally obtained by yaffe and saxel at the weizmann institute of science in israel in 1977. C2c12 myoblastoma cell differentiation and proliferation. A reducing redox environment promotes c2c12 myogenesis. Feb 15, 2014 when cultured in 8% serum during differentiation on uncoated surfaces to reproduce typical culture conditions for c2c12 cells, levels of mir1, mir3a, mir206, and mef2c were. Treatment with bone morphogenic protein 2 bmp2 cause a shift in the differentiation pathway from myoblastic to osteoblastic. Hyclone, usa, containing 10% fetal bovine serum with 1% penicillinstreptomycin gibco, usa, in 5% co 2 atmosphere at 37 c. The differentiation of c2c12 cells to myotube by paraquat. C2c12 mouse myoblast to osteoblast biomanufacturing.
After 96 hours, c2c12 cells were stained to detect mhcexpressing myotubes figure 1a1d, 1i1l and nuclei figure 1e1h, 1m1p. Nov 26, 2018 although primary cultures are more suitable for investigating in vivo skeletal muscle growth and differentiation as they represent a model that is closer to in vivo situations and clinical applications, serum free media have only been investigated on cell lines like c2c12 in terms of myogenic differentiation 16, 17. Bone morphogenetic protein2 converts the differentiation pathway of c2c12 myoblasts into the osteoblast lineage published erratum appears in j cell biol 1995 feb. Although primary cultures are more suitable for investigating in vivo skeletal muscle growth and differentiation as they represent a model that is closer to in vivo situations and clinical applications, serum free media have only been investigated on cell lines like c2c12 in terms of myogenic differentiation 16, 17. The c2c12 in vitro skeletal muscle line is a common, wellknown invitro model that is used to observe differentiation of skeletal muscle cells and assessment of the contractile forces of the differentiated cells li et al. 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. To investigate the effects of androgens and anabolic steroids on the proliferation and differentiation of c2c12 cells, the cells were seeded in 24well plates either in a regular or in a differentiation medium and treated with 10. At day 3, this increase was attenuated when the cells were treated with ccg1423 p c2c12 cells induced to differentiate by serum deprivation and treated in differentiation medium with 100. Metformin was refreshed every 24h and the levels of phosphop38 and total p38 protein were analyzed in crude cell protein extracts by sds page.
Differentiation of skeletal muscle and integration of myotubes with silicon microstructures using serum free medium and a synthetic silane substrate. Developed for in vitro studies of myoblasts isolated from the complex interactions of in vivo conditions, c2c12 cells are useful in biomedical research. The differentiation of these cells in vitro requires a switch from a serumrich medium to a less rich medium after the cells have reached confluence. Differentiation markers of mouse c 2 c 12 and rat l 6. Press start in the program and record the contraction sarcomere length and fura ratio. Thats why one has to use low amounts of adult serum because they are less abundant in. Differentiating c2c12 cell cultures contain a subpopulation of. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that c2c12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. Differentiation in c2c12 myoblasts depends on the expression of. For differentiation, cells were plated on gelatincoated tissue culture plates 0. C2c12 and l6 myoblasts were differentiated in both serumcontaining and serumfree. Skeletal muscle cell proliferation and differentiation are tightly.
C2c12 differentiation was induced at 80% confluence by switch ing media to dmem supplemented with 2% volvol horse serum hs. Currently, c2c12 murine myoblast cells are used to study myo. When cultured in 8% serum during differentiation on uncoated surfaces to reproduce typical culture conditions for c2c12 cells, levels of mir1, mir3a, mir206, and mef2c were. Our results showed that bmp7 expression was increased during c2c12 cell differentiation fig. When the myoblasts were 7080% confluent, c2c12 myoblasts 7. Positive and negative regulation of myogenic differentiation.
Two commonly used myogenic cell lines, mouse c2c12 and rat l6, usually differentiate at a low concentration of horse serum. Lipofectamine transfection of c2c12 cells bridges lab. In this study we compared the effect of horse serum with a medium containing a low percentage of ultroser g and rat brain extract. However, this is in stark contrast to results obtained with chick, rainbow trout, and mouse myosatellite cells also known as muscle stem or satellite cells in which myostatin inhibits proliferation and, as a consequence, initiates terminal differentiation 9. Myogenic differentiation is initiated upon reaching confluence by switching the cells to medium containing 2% horse serum supplemented with insulin. C2c12 cells are derived from the mouse skeletal muscle c2. Frontiers overexpression of striated muscle activator of. Myogenic differentiation was induced in a lowserum differentiation medium. Mef2a expression program during myogenesis in mouse c2. The c2c12 cells derived from murine skeletal muscle cells is a wellestablished model to study muscle regeneration and differentiation 15. However, the differentiation process of c2c12 is tedious and difficult to learn. The beneficial effects are likely to derive from pleiotropic interactions. Conditions that promote primary human skeletal myoblast. Comparison of initial stages of muscle differentiation in rat and.
Knockdown of hes6 mrna levels by sirna has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating c2c12 myoblasts, but factin filament formation is disrupted and both cell motility and myoblast fusion are reduced. Stars increases c2c12 skeletal muscle cell differentiation. The serum free medium used was one that we developed specifically for c2c12 cell growth and differentiation on fibronectin substrates, and enabled us to assess the affect of the ecm on myoblast proliferation and differentiation in the absence of the confounding variables present in serum. The c2c12 cell line differentiates rapidly, forming contractile myotubes and producing characteristic muscle proteins. Cyclic sinusoidal stretch was applied continuously for 14 days after differentiation in 8% and 2% serum. The striated muscle activator of rho signaling stars is an actin binding.
Damage to the myogenic differentiation of c2c12 cells by. The problem is that this cells grow like crazy, so if i want to transfect them and then induce the differentiation for some days the control is already too grown. The key to differentiating c2c12 is to switch them from a proliferative to a differentiating state. From day 3 onward, serum was decreased from 10% to 2% and changed to horse serum to initiate cell cycle exit and myogenic differentiation ie, serum depletion 12. These cells could be used for biomedical research and cell biology, especially of mammalian myoblast cells.
Cell growth protocol and differentiation treatment for the. In order to differentiate c2c12 cells, you have to slow down the cells by withdrawing them from proliferation and switch to differentiation state. For immunofluorescence, c2c12 cells were grown on 18mm coverslip to 10% confluency with dmem supplemented with 10% fetal bovine serum and myogenic differentiation was induced by serum withdrawal. S1, serum starvation medium triggered myogenic differentiation as indicated by formation of myotubes after 4 days of induction, while. Hes6 is required for actin cytoskeletal organization in. Creatine cr is a nutritional supplement promoting a number of health benefits. In accord with this notion, we previously demonstrated that multiple pleiotropic effects, including. During the process of c2c12 myoblast differentiation, we. After 24 h of culturing, c2c12 cells were maintained in serum free medium and then treated with different concentrations of tgf. We previously found that 10% cyclic mechanical stretch at 0. After 96 hours, cells were stained with antibodies against mhc ad or with hoechst 33342 eh.
C2c12 mouse myoblasts were maintained in dmem containing 20% fbs and 0. Differentiation medium dm, consisting of 1% penicillinstreptomycin and 2% horse serum gibco, usa, replaced growth. And when these cells overgrown they differentiate as well. For differentiation, we switch to 5% horse serum, 1x penicillinstreptomycin in dmem while c2c12 myoblasts is about 6080% confluent. Maged1, a new regulator of skeletal myogenic differentiation and. C2c12 cells were plated on glass slides for immunofluorescence and cultured for additional 120 hours in dm. C2c12s are commercially available but because variable handling of this line can select for cells with different kinetics or poor differentiation performance, the wold lab will provide plugs of these c2c12s upon request. The localization of hes6 protein changes during differentiation, becoming predominantly nuclear. Why does horse serum induce differentiation of c2c12. When c2c12 cells reached 90% confluence, the medium was switched to differentiation media containing 2% horse serum to induce myogenic differentiation. Sparcl1 promotes c2c12 cell differentiation via bmp7.
During normal culture using fbs the cells are highly proliferative because fbs contains factors for proliferation. Direct differentiation into osteoblast using bone morphogenic protein 2 bmp2. A new cellbased assay to evaluate myogenesis in mouse. Immunofluorescence analysis of the differentiation marker, myosin heavy chain, myhc, in c2c12 cell line cultured in gm or dm. C2c12 cells were differentiated in culture medium containing horse serum.
Jul, 2018 effect of hs on c2c12 cell differentiation. C2c12 myoblasts and 10t12 fibroblasts were maintained in growth medium gm consisting of dulbeccos modified eagles medium supplemented with 10% fetal bovine serum. Grow myoblasts in 100 mm plates to 50% confluence maintain cells at 37c and 10% co 2 in growth medium dulbeccos modified eagles medium dmem, supplemented with 20% fetal bovine serum fbs and 1% penicillinstreptomycin antibiotic. The master regulator gene prdm2 controls c2c12 myoblasts. The differentiation grade of cells in culture is dependent on the composition of the culture medium. For proliferation assay, c2c12 were grown or not with 1. Early studies using c2c12 myoblasts, an immortal cell line derived from dystrophic mouse thigh muscle 4, suggest that recombinant myostatin generated in bacteria inhibits both proliferation and differentiation 5,8. Vitamin d signaling regulates proliferation, differentiation. Myogenic differentiation in vitro has been usually viewed as being negatively controlled by serum mitogens. Fv10i confocal laser scanning microscope with dedicated software at a 150x. C2c12 has been tested negative for mousepox ectromelia virus. Serum deprivation induces myogenic differentiation of c2c12 cells. The mammalian target of rapamycin regulates c2c12 myogenesis. Research paper mir696 regulates c2c12 cell proliferation and.
Panax ginseng total protein facilitates recovery from. We have compared several serumfree media for the differentiation of c2c12 myoblasts and assessed the extent of differentiation in several ways including as. Assessing differentiation potential of c2c12 myoblastic. Dexamethasone treatment at the myoblast stage enhanced. Research paper fibroblast growth factor 9 fgf9 inhibits myogenic differentiation of c2c12 and human muscle cells jian huang a, kun wangb, lora a. Working with the c2c12 cell line research in myogenesis. Fgf9 as low as 10 ngml inhibited myogenic differentiation, suggesting that fgf9 might be a potential inhibitory factor produced from bone cells with effects on muscle cells. Skeletal muscle differentiation is a multistage highlyregulated process that includes myoblast division, elongation, and fusion 5. C2c12 transfection reagent mouse myoblast cells altogen. C2c12 and l6 cells differentiated in serumcontaining and serumfree. I am working with c2c12 cell line, using medium with 2% horse serum to induce differentiation. Myoblast cell lines are grown and differentiated readily in cell culture.
Cultures were checked for myotube formation, the activity of creatine phosphokinase and the presence of sarcomeric actin. In this study, we test the effects of a serumfree medium on both c2c12 cells and l6 cells to determine whether one medium could induce differentiation in both of these model systems. C2c12 differentiation and transfection cell biology. C2c12 myotubes express contractile proteins and, when left to differentiate for an extended period of time, can spontaneously contract. Culturing c2c12 myotubes on micromolded gelatin hydrogels. C2c12 induced to differentiate by serum deprivation in differentiation medium. Regulation of myoblast differentiation by metabolic. Research paper dexamethasone treatment at the myoblast stage. Mouse c2c12 cells were induced to differentiate to muscle by changing from. In fact, c2c12 cells are immortal, proliferate rapidly, and will not differentiate unless induced. Lipofectamine transfection of c2c12 cells bridges lab protocols.
In c2c12 myoblasts, myod is expressed but is inactive when cells are. C2c12 cells were maintained in dulbeccos modified eagle medium containing 10% fetal bovine serum at 37c, 5% co 2. C2c12 and l6 myoblasts were differentiated in both serum containing and serum free media. Dc200 with a 20x objective, and recorded using imi leica software. Two cell lines typically used for investigating the growth and differentiation of muscle are the mouse cell line c2c12 and the rat cell line l6. Serum stimulation 20% fbs increased nuclear mrtfa levels in c2c12 myotubes at differentiation day 3 p differentiation day 5 supplementary figures 2h,g. To induce cell differentiation, culture medium was switched to differentiation medium dm with 2% horse serum and 1% penicillinstreptomycin gibco, usa. Not all myoblast cell lines have the same requirements for growth and survival. C2c12 s are commercially available but because variable handling of this line can select for cells with different kinetics or poor differentiation performance, the wold lab will provide plugs of these c2c12 s upon request.
Dexamethasone treatment at the myoblast stage enhanced c2c12. To prevent differentiation, cells were only grown to 80%. C2c12 differentiation and transfection posted in cell biology. In this study, we test the effects of a serum free medium on both c2c12 cells and l6 cells to determine whether one medium could induce differentiation in both of these model systems. Pdf differentiation of myoblasts in serumfree media.
C2c12 differentiation was induced upon 70% confluence by serum withdrawal dmem with 2% horse serum hs, invitrogen. Skeletal muscle cell proliferation and differentiation are tightly coordinated by a continuum of molecular signaling pathways. When exposed to low serum differentiation media, these cells differentiate and fuse into a multinucleated syncytium referred to as a myotube. C2c12 cells have 2 phases proliferation and differentiation. Myostatin reduces akttorc1p70s6k signaling, inhibiting myoblast differentiation and. A, at day 7, there were notably fewer myotubes in c2c12 cells treated with 1,25oh 2 d. Myod and myogenin protein levels were analyzed by western blotting either in c2c12 cells maintained in dmem containing 10% fcs growth condition, or 24 hours and 48 hours after switching cultures to dmem supplemented with 1% hs differentiation condition. After 72 h, cells were fixed in 4% paraformaldehydephosphate buffered saline pbs for 10 min at room temperature and incubated in icecold. In our study, c2c12 cells were used to investigate the. Here we studied the effect of paraquat on the differentiation of mouse myoblast cell line c2c12 cells to myotube. Any suggestions for a recipe for c2c12 differentiation medium.
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