A role for protein phosphatase 2alike activity, but not. Research article open access the proteasome inhibitor. Mouse c2c12 myoblasts american type culture collection, blau et al. G, but the active tension generated by c2c12 cells that differentiated in ult. Culture, differentiation and transfection of c2c12. Figure 2ac show the nuclear, cytoplasmic and mitochondrial fractions obtained from a single starting sample from each of the different tissue cell types. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. We have shown previously that palmitate treatment of c2c12 skeletal muscle myotubes causes inhibition of the protein kinase b pkb pathway and hence reduces insulinstimulated glycogen synthesis through the elevation of intracellular ceramide levels. C2c12 cells were cultured on collagen type icoated dishes using dmem. This protocol explains the basic procedures of culture, transfection and differentiation of c2c12 myoblast cells. 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. Effects of a combined mechanical stimulation protocol. Atcc crl1772 were maintained as exponentially growing myoblasts in a growth medium consisting of dulbeccos modified eagles medium dmem supplemented with 20% heatinactivated fetal calf serum, 4 mm lglutamine, 100 iu. Ceramide is known to activate both atypical protein kinase c apkc.
Cells that are difficult to detach may be placed at 37c to facilitate dispersal. Cell culture and treatment c2c12 cells were cultured incomplete proliferation medium consisting of dmem supplemented with 10% heatinactivated fetal bovine serum fbs, 100 iuml of penicillin, and 100. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Developed for in vitro studies of myoblasts isolated from the complex interactions of in vivo conditions, c2c12 cells are useful in biomedical research.
C2c12 myoblasts are commonly used in biomedical laboratories as an in vitro system to study muscle development and differentiation. C2c12 cells atcc crl1772 were differentiated for 5 days and then disassociated and fixed in bd cytofix fixation buffer cat. All further steps are described in the subculture section. Constitutive expression of p67 increases cpkcalpha level.
Prdx6 plays a main role in the crosstalk between aging and. A very valuable cell line to study bmp signal transduction is the murine premyoblast cell line c2c12. Rock the culture platedish gently to distribute the particles evenly across all the cells. Add appropriate aliquots of the cell suspension to new culture vessels. Uniaxial cyclic strain of human adipose derived mesenchymal stem cells and c2c12 myoblasts in coculture james m dugan1,2, sarah h cartmell 1 and julie e gough abstract. Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e. C2c12 cells are easyto culture highly proliferative cells, display low basal bmp signaling activity and good bmp responsiveness, and can be transfected with high efficiency using most transfection reagents. Cells were grown in 100mm petri dishes becton dickinson, franklin lakes, nj and cultured in minimal essential media mem containing 10% bovine calf serum, penicillin 100 uml, streptomycin. Add the magnetic particlesvirus mixture dropwise to the cells to be transduced.
Place the culture platedish on top of the magnetic plate i. Preparation and culture of myogenic precursor cells. Cell culture guidelines the following is a general guideline for culturing of cell lines. Dose and time dependent effect of dex on c2c12 myoblast cell line was analyzed at myoblast and myotube stage, respectively. Release of glutaraldehyde from an albuminglutaraldehyde. Recommended transfection protocols for 24well plate.
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. The c2c12 mouse myoblast cell line was purchased from the american type culture collection manassas, va and used for all studies. None effect on aging andor sarcopenia incidence have been reported among these two strains. In c2c12 myoblasts, the expression and activity of cpkcalpha is very high and this decreases during the course of differentiation of myoblasts into myotubes and ultimately myofibers 810. Culture conditions for hskm are different from those that enable rapid c2c12 myoblast differentiation. Satellite cells of muscle from old mice and humans have also been shown to undergo senescence resulting in their reduced functionality20. Add required amount of prewarmed cell culture media to fresh flask.
The cell culture flasks are completely filled with cell culture medium to prevent loss. Chromosome conformationcapture technologies are widely used in 3d genomics. Mammalian cell tissue culture techniques protocol abcam. Assessing differentiation potential of c2c12 myoblastic cells on. Research paper dexamethasone treatment at the myoblast. C2c12 cell cultures and at muscles using the protocol described in figure 1, we report the mean fraction yield values obtained from replicate experiments n5 table 1. Working with the c2c12 cell line research in myogenesis. Differentiation was initiated by replacing the cell growth medium. This kit has been determined to be optimal for transfecting following cell types. A significant amount of studies have also used murine primary myoblasts22. After incubation for 100 minutes, the mrc5 cells were stained with carbolfuchsin, and the c2c12 cells were assessed for viability using the 34,5dimethylthiazol2yl2,5diphenyl. Culture, differentiation and transfection of c2c12 myoblasts. Can anyone suggest a protocol for c2c12 cell differentiation to myotubes.
Exonuclease combinations reduce noises in 3d genomics. The level of differentiation was determined by myh1, pax7, atrogin1, and myostatin mrna expression and fusion index. To induce insulin resistance, c2c12 cells were cultured in dmem containing 15 gliter of glucose, 10% fetal bovine serum, sodium pyruvate, 1 gml of insulin, 100 nm dexamethasone, and 1%. Cell culture c2c12 cells were maintained in dulbeccosmodi. C2c12 mouse myoblasts are the excellent model system to study the details of myogenesis. C2c12 myoblasts american type culture collection, va, usa were cultured at 37 c in 5% co2 in gm. Medium was changed once at 24 h to account for hydrogen peroxide hydrolysis. Cells were cultured in dulbeccos modified eagles medium dmem. The cell line c2c12 is an immortal line of mouse skeletal myoblasts. C2c12 cell culture, differentiation treatment, and crosslinking protocol. The most established cell line enabling the study of muscle homeostasis is murine c2c12 cell line21. Cell growth protocol and differentiation treatment for the.
Develop a highthroughput and reliable method to determine stiffness of softwell cell culture plates. Cell culture and differentiation protocol mouse myogenic c2c12 cells american type culture collection. C2c12 cells were transfected with vector of vamp2 wildtype wt or mutant mut, then cultured for 72 h of proliferation. In short, the culture medium was collected from cells after stretching for 24 or 48 hours as well as from unstretched controls, filtered through a. Can anyone suggest a protocol for c2c12 cell differentiation to. Continuous mild heat stress induces differentiation of. Product information c2c12 cells cls cell lines service. Lipopolysaccharide and proinflammatory cytokines stimulate. Culture on a growth factorreduced matrigel gfrmgcoated surface in 2% equine serumsupplemented differentiation medium to promote hskm differentiation under static conditions was compared with culture conditions used for c2c12 cell. Tgf regulates collagen type i expression in myoblasts and. Culturing and differentiating c2c12 cells bridges lab.
If the density of the culture is not even, split it sooner than usual or only 1. Methods for reducing cell growth edge effects in agilent. Cell culture the mouse myogenic c2c12 cell line was obtained from atcc and cells were used up until passage number 8. Cultures must not be allowed to become confluent as this will deplete the myoblastic population in the culture. Description myoblast cell line derived from thigh muscle of c3h mice after crush injury.
Culturing c2c12 myotubes on micromolded gelatin hydrogels. C2c12 are very tricky cell line, you must keep cells in culture at confluence less then 60%, otherwise you will select. C2c12 myoblastoma cell differentiation and proliferation. The culture medium for the c2c12 cells was replaced with fresh serumfree medium supplemented with 10% volvol supernatant. Opg immunoassays the mouse opg immunoassay was performed according to the manufacturers protocol rd system.
Which is the best protocol to differentiate c2c12 cells. The microrna1273p directly targeting vamp2 in c2c12. Split cells normally 110 or 120 into 10% fbs in dmem with 1x psg. During skeletal myogenesis, genomic reprogramming toward terminal differentiation is achieved by recruiting chromatinmodifying enzymes to musclespecific loci1,2. Skeletal muscle is an appealing topic for tissue engineering because of its variety in applications for regenerative medicine, in vitro physiological. The mouse myogenic c2c12 cell line was obtained from atcc and cells were used up until passage number 8. I have seen several different protocols but didnt try. Protein extract ripa recipe from xiping zhang at esser lab. The differentiation medium contained 3% horse serum instead of 10% fs10. C2c12 cell culture the c2c12 mouse muscle myoblast cell line atcc crl1772 was obtained from atcc wesel, germany. Technical note open access a simple protocol for the. C2c12 and human skeletal muscle cell culture conditions c2c12 myoblasts were cultured following our own previously published protocols 3,44. Active tension generation was observed for cells that differentiated in aim and ult. Product overview the cell line nucleofector tm kit v is one of our five nucleofector tm kits suited for transfecting cell lines in the nucleofector tm 2b device.
Conditions that promote primary human skeletal myoblast. Cell culture and differentiation murine skeletal muscle cell line c2c12 was purchased from american type culture collection manassas, va, usa. The base medium for this cell line is atccformulated. The cells readily proliferate in highserum conditions, and differentiate and fuse in lowserum conditions.