Myoblast differentiation marker

S.A.M.

S.A.M.

uniquely dreadful
Valued Senior Member
I'm running some muscle cell culture studies using L6 and C2C12 cell lines. I am using Dulbecco's modified Eagle's medium with 10% fetal bovine serum as a growth medium and DMEM with 2% Horse serum as a differentiation medium.

Since I would like to treat the cells only after they are fully differentiated, but as soon as possible post differentiation, I am looking for an easy marker of differentiation to optimise my protocol.

The markers I have already come across are:

1. myotube formation: microscopy (too subjective)
2. creatine phosphokinase activity: using activity assay kit ($$$)
3. sarcomeric actin production: using dot blots (time consuming)

Anyone know of any specific staining method or a faster ATM assay for determination of differentiation status?
 
S.A.M. said:
I'm running some muscle cell culture studies using L6 and C2C12 cell lines. I am using Dulbecco's modified Eagle's medium with 10% fetal bovine serum as a growth medium and DMEM with 2% Horse serum as a differentiation medium.

Since I would like to treat the cells only after they are fully differentiated, but as soon as possible post differentiation, I am looking for an easy marker of differentiation to optimise my protocol.

The markers I have already come across are:

1. myotube formation: microscopy (too subjective)
2. creatine phosphokinase activity: using activity assay kit ($$$)
3. sarcomeric actin production: using dot blots (time consuming)

Anyone know of any specific staining method or a faster ATM assay for determination of differentiation status?
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Wow! I thought nutritionist just told one to stop eating fat and grimaced condescendingly at ones fried mars bar and chips.
 
GhostofMaxwell. said:
Wow! I thought nutritionist just told one to stop eating fat and grimaced condescendingly at ones fried mars bar and chips.
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Thats dietetics, nutritionists study the biological effects of nutrients on metabolism. My area of interest is chronic disease (namely obesity). :)
 
GhostofMaxwell. said:
On the flip side: Is there much study of too little fat being bad for you?
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Sure, especially for essential fatty acids. Leads to dry flaky skin and is accompanied by deficiencies of fat soluble vitamins which regulate growth and differentiation as well as cell survival.

Basically plays havoc with all actions that are regulated by fat or fat derived products, including sex hormones, prostaglandins, immune system, cell polarity; causing severe problems with eyesight, skin, nail formation, blood clotting, kidney function, bone growth and repair, reproductive functions, and cellular energy. Among others.

However since most of us eat more than enough fat, the only kind of lipid deficiency seen is that of essential fatty acids (those which cannot be synthesised by the body)

http://www.causeof.org/efa_d.htm
 
S.A.M. said:
Sure, especially for essential fatty acids. Leads to dry flaky skin and is accompanied by deficiencies of fat soluble vitamins which regulate growth and differentiation as well as cell survival.

Basically plays havoc with all actions that are regulated by fat or fat derived products, including sex hormones, prostaglandins, immune system, cell polarity; causing severe problems with eyesight, skin, nail formation, blood clotting, kidney function, bone growth and repair, reproductive functions, and cellular energy. Among others.

However since most of us eat more than enough fat, the only kind of lipid deficiency seen is that of essential fatty acids (those which cannot be synthesised by the body)

http://www.causeof.org/efa_d.htm
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Yeah I've heard about the Omega-3 and that..........But say you ignore the good fat's for sake of argument: You have one person who deep fries everything in the worst fats possible and one who boils everything and removes virtually all the fat from his diet. Which would be the worst scenario?
 
GhostofMaxwell. said:
Yeah I've heard about the Omega-3 and that..........But say you ignore the good fat's for sake of argument: You have one person who deep fries everything in the worst fats possible and one who boils everything and removes virtually all the fat from his diet. Which would be the worst scenario?
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Its all malnutrition.
 
S.A.M. said:
I'm running some muscle cell culture studies using L6 and C2C12 cell lines. I am using Dulbecco's modified Eagle's medium with 10% fetal bovine serum as a growth medium and DMEM with 2% Horse serum as a differentiation medium.

Since I would like to treat the cells only after they are fully differentiated, but as soon as possible post differentiation, I am looking for an easy marker of differentiation to optimise my protocol.

The markers I have already come across are:

1. myotube formation: microscopy (too subjective)
2. creatine phosphokinase activity: using activity assay kit ($$$)
3. sarcomeric actin production: using dot blots (time consuming)

Anyone know of any specific staining method or a faster ATM assay for determination of differentiation status?
Click to expand...

External morphological differentiation? I know a guy who said he used to do that.
 
GeoffP said:
External morphological differentiation? I know a guy who said he used to do that.
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I decided to go with the kit, I already started the experiment two days ago. Thanks

Its 8-12 days for full differentiation and I want the preliminary results in a month, so I could not wait.
 
S.A.M. said:
I decided to go with the kit, I already started the experiment two days ago. Thanks

Its 8-12 days for full differentiation and I want the preliminary results in a month, so I could not wait.
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You should see if you can scope them anyway. Could save $ later.
 
GeoffP said:
You should see if you can scope them anyway. Could save $ later.
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Oh I'm gonna do that anyway, but my experimental design is predifferentiation, during differentiation and post differentiation. Since I am using a differentiation media the first and second are no big deal, but the range of full differentiation varies and I was hoping to use a couple of 12-well plates to standardise the point of full differentiation. Trouble is differentiation is affected if you split the cells over 70% confluence (in later passage numbers that is) so I want to keep it as consistent as I can for both splitting and differentiation points.

Oh well, I'll probably have to depend on my stellar vision anyway, but I hate not having more standard options, especially when the treatment groups may run into several flasks and wells.:(
 
Interesting but I'm only a humble genotyper. What all that mean in normal-talk?
 
GeoffP said:
Interesting but I'm only a humble genotyper. What all that mean in normal-talk?
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I'm using 6-well plates for my experiment (bigger wells to get sufficient protein and RNA for immunoblotting and RTPCR)

cultw-plates-002.jpg


And 12 well plates for optimisation

cultw-plates-003.jpg


I have an experimental design over 3 weeks with three different time points and four treatment groups; three days per treatment.

Exponentially, with additional dishes to maintain cells for the next experiment, the number of wells multiplies, so I don't want to have to do many do-overs. I'd like to get the prelim data in a month for a grant due Nov.
 
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Confluence refers to density; myoblasts are adherent cells (stick to well or flask) and on reaching 100% confluence begin to change characteristics, taking on specialised features of muscle. We can use different treatments to modify these characteristics and study the effects of different nutrients on muscle mass, muscle fibre type and muscle proliferation (hypertrophy vs hyperplasia).

CRL-1458_mg1.jpg


Thats part of what I am planning to write a grant on; I need data for protein expression patterns with the addition of 'x' nutrient at different doses at different stages of muscle proliferation and differentiation.

We usually split the cells for further passaging (seeding other wells with cells to grow for different treatment groups, for storage of a certain passage number, for maintenance of cells for further experiments at a different passage number*) before they clump together (around 50% confluence or less) since once they get attached to each other, they start expressing specialised proteins. Then further passaging at this stage affects cell fusion in later passages.

*Passage number: every time the cells reach a confluence at which you split them (divide them for further growing), they pass one passage number. We usually don't use cells past passage 20, the results are not as extrapolative
 
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Wow S.A.M. that looks interestnig:)
i'm going to start in few weeks to work with L6 cells. I have no colegues working with this cells of give me some guidelines.
Right now im reading articles, looking for protocols and general knowledge about L6.
Could you give me any advice?

thanks!
 
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