Skeletal Muscle

Skeletal Muscle – Science topic

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Questions related to Skeletal Muscle
Syed Amir Gilani
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I heard a GP state that ultrasound was useless for tendons. As a specialist musculoskeletal sonographer recently returned to the UK, I found this comment quite disturbing. Are patients missing out on vital diagnosis and treatment simply because referrers think they need MRI access to solve cases?
 
Wangang Zhang
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As mentioned in the title. Could anybody help to provide some references about these questions?
 
The report about the free calcium concentration in postmortem muscle is not consistent, but it is sure that the free calcium will increase in postmortem muscle due to the function loss of proteins regulating calcium retake. In living animals, the calcium concentration would be around 100 nM for relaxation and at 5 uM for contraction of skeletal muscle. It can inrease to 100-200 um in postmortem muscle.
A researcher
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I have used uranyl acetate in 50% ethanol for 10 followed by a wash in 50% ethanol then followed by 10 min in lead citrate in boiled water. I can't seem to get definition of the sarcomere.  Muscles were here fixed in 2.5% glutaraldehyde then 1% Osmium tetroxide. 
 
please check this video https://www.youtube.com/watch?v=403ruZpNw2k
Daniel Lark
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I am looking at mitchondrial function in mouse skeletal muscles. What genes would be the best markers to show mitochondrial function/biogenesis/content?
 
As Andrea said, PGC-1a would be best for biogenesis.  For measuring mitochondrial content, you could measure mtDNA or citrate synthase activity.  I think the best indication of function could be enhanced from in frozen tissue is measuring OXPHOS complexes at the protein level.  Abcam sells a great antibody that includes all five: http://www.abcam.com/total-oxphos-rodent-wb-antibody-cocktail-ab110413.html
Anya Snary
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My current estimated tg time is 13 hours, the equation I have to complete is  Log NT  =  Log No  +  Log 2/tg X  T Current values I have are: 4.954 = 4+ (0.301/13) x 24  in log 10 values However, I understand my second line of my equation needs to equal 0.954 however I am struggling to see how to do this - any tips? 
 
Shabir Kumar Anant
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We want to use FK506 to disrupt binding between RyR1 and FKBP12 in whole skeletal muscle lysates. Does anyone have some experience in regard to the concentration of FK506/ug Protein in the lysate? Is there something that should be considered in regard to the cell lysis buffer (e.g. pH, ion strength, detergent, etc.)? Routinely we use the cell signaling cell lysis buffer. Thank You for your help! Daniel
 
no dear
Mark E Cleasby
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I am going to run a BCA assay (ThermoScientific) with rat skeletal muscles (soleus & EDL) to determine protein concentration. Since I have used RIPA to homogenize tissues (10:1), I won't use the Bradford protocol (overestimation). What is a proper ratio for working reagent to sample for the plate reader method? I have seen some muscle papers that work at a 1:50 and 1:100 ratio, but the product insert shows a 1:8 and 1:20 ratio. Is there an optimal ratio for this protocol? Please, and thank you.
 
For a similar ratio of rat muscle to RIPA buffer, we find that a 1:50 dilution of the lysate in water is compatible with both BCA and Bradford assays and gives reproducible results within the manufacturer's recommended range of BSA standard dilutions. For the Pierce BCA assay, this involves 25ul of diluted lysate and 200ul of reagent.
Bula Bhattacharyya
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I am interested in the effects of aging on the properties (e.g. physiology, histology, morphology and function) of skeletal muscles, pain perception, and central pain processing. Please recommend some classical and must-read materials at your convenience. Thank you very much.
 
You can read this article Eur J Pharmacol. 1994 Mar 11;254(1-2):97-104. Age-induced alteration of neuromuscular transmission: effect of halothane. Bhattacharyya BJ1, Tsen K, Sokoll MD
Kozina Zh. L.
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The amount of 3-methyl histadine per gram of skeletal muscle is a constant. Therefore, if there are issues with methylation, one would expect that this must limit the amount of myofibrillar protein that can anabolically be synthesised. Does anyone know if this concept is, in fact, true?
 
The above assumption is logical. However, the literature is not enough data to draw conclusions. It is possible to partially solve the problem of muscle growth with a lack of methylation Supplementing the necessary amino acids.
Manoj Balakrishna Menon
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I would like to detect a phosphorylated protein by immunofluorescence in skeletal muscle sections. Is there any adaptation of the protocol specific for phosphorylated proteins, use of phosphatase inhibitor or something else? A recommended protocol to use?
 
if I understand your question correctly, u have experience with immunofluorescence with tissue sections and would like to do phospho-antigen staining for the first time. I do not think there are any special general protocols or precautions for phospho-staining and the success and staining protocol will depend completely on how good your antibodies are. Some tissies could be ex-vivo treated with phosphatase inhibitors, but this will not be helpful if you are interested in the  in-vivo situation. https://www.bd.com/resource.aspx?IDX=17718 If you have the right antibodies, go ahead with standard protocols with good controls ( like co-incubation with phospho/non-phospho-peptide- immunogens sold by many companies, phosphatase treated sections etc.). If you are using a new uncharacterized antibody, it will be easier to study its specificity (target and phospho-specificity) in cell lines before using it in tissue sections. Good Luck.
John Hildyard
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I have a student who is comparing exercising rats (that had voluntary access to running wheels for 8 weeks), sedentary rats (that were in wheel-free cages for 8 weeks), and de-trained rats (that had running wheels for 4 weeks and then were sedentary for 4 weeks). She will be doing qPCR on the soleus muscle for VEGFA, FGF2, and MUSTN1; and the housekeeping gene they are considering is ACTB. She would like to use more than one reference gene to increase the validity of the assay so I said I'd ask the Research Gate community to see if anyone has any other suggestions.
 
ActB is pretty terrible in muscle, in my experience (though caveat: I work with mouse muscle).  For mouse muscle I generally use Pak1ip1, Ap3d1 and GAPDH (from the primerdesign genorm-plus kit), but 18S is also not too bad. I would recommend you use at least two reference genes (ideally three) and if you have the time & money, I'd suggest you test a panel of 6-10 candidate genes (against a subset of samples from your test and control groups) then run the numbers through geNorm, normfinder or bestkeeper to identify suitable genes.
Wangang Zhang
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Do anyone have an idea about PEDF secretion from skeletal muscle.
 
Dear Revathy, This study provided some data about the concetration of PEDF from skeletal muscles. http://ajpendo.physiology.org.sci-hub.org/content/301/5/E1013.short
Maximilian Kleinert
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In liver Akt seems to be crucial for normal Glucokinase (ie Hexokinase IV) expression. I am wondering if the same holds true for muscle and Hexokinase II. Is anybody aware of Akt KO studies or similar that have assessed the impact on HKII in muscle? Thank you for your help.
 
Thanks, Dudley.
Isabel Punzon
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I need to insert a gen systemically, but with the skeletal muscle as specific target. Is the adeno-associated virus type 6 (AAV6) vector a good vehicle? Do you know other options?
 
In humans and dogs the AAV6 can be blocked by the G3BP, not in mice...Maybe it's better the AAV9. Take a look at his paper: Human Galectin 3 Binding Protein Interacts with Recombinant Adeno-Associated Virus Type 6. Denard et al. 2012. Journal of Virology ; June 2012 Volume 86 Number 12
Martin Ward Platt
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I'm going to measure the oxidation rate of palmitate in vitro by using a skeletal muscle model with C2C12 cell. I have read many papers but  yet have not been able to find out a suitable method avoid using radioisotope 14C. Is there any possible method available. Appreciate any respond.  
 
This is a curious question. C14 is cheap, simple and well validated.  The only alternative is to use a C13 (stable isotope) label, which is feasible but much more complicated and expensive.  C14 methods only need an emission counter; stable isotope methods need mass spectrometry.  I would ask: why do you need to avoid C14?
Wojciech Pokrzywa
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I want to measure ubiquitination following IP of my proteins of interest. Does anyone have experience with ubiquitin antibodies that work well in skeletal muscle tissue or L6 muscle cells?
 
They are quite specific, but of course depends on the protein amount,success of your IP etc. We performed IP from C2C12 cells, detection with anti-Ub 1/5000 in 3 milk PBS-T, let say over night. Secondary 1/10000, 45min.  Check the conditions firstly on your input,before IP. For us 25uq of proteins in the lane was quite enough to see a polyubiquitylation patterns. 
Paul K Canavan
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I need a reference that explains if a dominant arm  activates more than the non-dominant one during an exercise or in static conditions.
 
This article below may help; Handedness: Dominant Arm Advantages in Control of Limb Dynamics Leia B. Bagesteiro , Robert L. Sainburg Journal of NeurophysiologyPublished 1 November 2002Vol. 88no. 5, 2408-2421DOI: 10.1152/jn.00901.2001
Faranack Nader Benz
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I wonder about the need of MVC record if I'm going to register EMG signal during 60 m race walking trials. Is there a better method to normalize muscle activity while performing such dynamic movements?
 
using comparative accessment of pre- stress and post- stress dynamics is a convenient instrument for variety of diognosis.
Marco Brotto
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I want to do some experiments in cells deficient in zinc, but I only could find protocols in other cell types (for example endothelial cells). 
 
Have you thought of conducting the skeletal muscle cells culture as you normally do and then adopting the endothelial protocols to the muscle cells?
Bradley T Elliott
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I am doing western blot for skeletal muscles samples. I tried different antibodies (beta-actin, alpha-actin & alpha-tubulin) for housekeeping protein (HKP) (control). The problem is some of the samples do not express the HKP but my target proteins were expressed in all my samples. Example, out of 8 samples, only 4-5 samples expressed the HKP. Please can anyone help and explain what could have caused this problem. I will also appreciate suggestions on the antibody I will use. Thanks
 
I would strongly second the comment by Nicolas. Our group normalizes to Ponceau S after transfer, in place of commassie, as recomended by this paper (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3809032/). We've found them to be more sensitive to changes in total protein loaded than 'housekeeping' proteins.
Cleber Ferraresi
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I am having some problems to normalize my qRT-PCR results using GAPDH because of its low expression at day 3 post-cardiotoxin injection in mice. I also tried to check the gene expression of several other housekeeping genes (HPRT, TBP,  RPL7, Cyclophilin B and 18S (very low Ct!) during regeneration but they all vary between samples. Can anyone suggest another housekeeping gene with a stable expression in cardiotoxin-injected muscles? Thank you
 
Hi Pamela and Mohamed,  I tryed these genes as housekeeping for skeletal muscles: GAPDH, B-actin, HPRT1, RPS29, RPS13, RPS20 and RPL27. The most stable genes were RPS20>RPS13>RPL27>RPS29>HPRT1>B-actin>GAPDH
Giacinto Libertini
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1. About the term of " cardiac sacropenia", what is current common sense about the association of sacropenia and aging heart (or other term such as cardiac sacropenia?) 2. Another question is about asian population, What is any reference about appendicular skeletal muscle mass
 
The correct term is “sarcopenia” and means cellular atrophy of muscle cells. For question1: Sarcopenia must be seen in the context of aging phenomenon. Please, see: Libertini G., Programmed aging paradigm: how we get old. Biochem (Mosc) 2014, 79(10):1004-16. (If you want a free copy, ask me giacinto.libertini@tin.it) In this paper, about cardiac sarcopenia: “An old and deep-rooted belief is that the heart is an organ incapable of regeneration and without cell turnover. On the contrary, “The Heart is a Self-Renewing Organ” [13]: in a normal heart, every day about 3 million myocytes die by apoptosis and are replaced by cardiac stem cells: “the entire cell population of the heart is replaced approximatively every 4.5 years … The human heart replaces completely its myocyte population about 18 time during the course of life, independently from cardiac diseases.” [13]. Cardiac stem cells duplicate and differentiate, allowing myocyte turnover, and show age-related telomeric shortening and cell senescence [36-38]. In the old heart there is a global loss of myocytes, with a progressive increase in myocyte cell volume per nucleus [39]. The decreasing number of myocytes is due the progressive decline in the ability to duplication of cardiac stem cells [13]. The decline of cardiac contractile capacities causes an enlargement of the heart that conceals the underlying atrophy of the contractile cells. So, in apparent contradiction, the heart chambers are dilated and the senile heart, although atrophic as number of cells, is morphologically hypertrophic [40]. “With aging, there is also a progressive reduction in the number of pacemaker cells in the sinus node, with 10 percent of the number of cells present at age 20 remaining at age 75. ... Age-associated left ventricular hypertrophy is caused by an increase in the volume but not in the number of cardiac myocytes. Fibroblasts undergo hyperplasia, and collagen is deposited in the myocardial interstitium.” [40] The heart shows “... some increase in the amount of fibrous tissue and fat in the atrial myocardium with a decrease in the number of muscle fibres, and loss of fibres in the bifurcating main bundle of His and at the junction of the main bundle and its left fascicles, with lesser degrees of loss in the distal bundle branches.” [41]. Drugs effective in “organ protection”, as ACE-inhibitors, sartans and statins, are effective in the prevention of atrial fibrillation [42, 43].”
Alexander Streng
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See above
 
I am guessing you are looking at Troponin T. That protein is highly expressed in muscle tissue and is about 30-35 kDa. But it is impossible to be sure without any identification experiments. Try blotting it with a skeletal troponin T antibody. Troponin T is very well preserved in its centre, so you may get lucky. Your best bet at identifying the protein is to cut it out and do a digest+mass spectrometry, if you have access to those techniques.
Christopher Turner
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Glycogen-autophagy is known to occur in skeletal muscle. What is the fate of glucose resulting from glycogen-autophagy in skeletal muscle?
 
I suspect that glucose is trafficked from the endosome-lysosome system into the cytosol. It is then metabolised through the pentose-phosphate pathway, trafficked through to the Kreb's or converted to glycogen. 
Daisy Motta-Santos
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What type of skeletal muscle was used in this study to identify VAChT in the motir end plates?
 
A researcher
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Please can anyone suggest the incubation time required for Insulin for studying the downstream phosphorylation of its substrate proteins of mouse and human skeletal muscle (C2C12 and HSMM)?
 
It depends on dose you are using. I usually treat C2C12 myoblast with 100nM insulin for 15 minutes.
A researcher
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Skeletal muscle calcifications as possible results of acute myositis
 
please have a look http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493084/
A researcher
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Could you please identify these tissues?  Thanks 
 
1 and 5 are longitudinal sections of skeletal muscle  they show clearly striations
Martino V. Franchi
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what is reliable and valid method of measuring hamstring muscle length? objectively. as tool used in research to measure the outcome of interventions used for increasing hamstring length or flexibility.
 
Dear Shakil, Firstly, I totally agree with Professor Lieber. You need to be more specific on what you are looking for. However, If interested in the effects of exercise (and different types of contractions) on adaptations in muscle fascicle length, I suggest you read first about muscle architecture significance (in terms of how changes in muscle fascicle length do influence muscle function - Professor Lieber's  extensive review is attached); then, there is some work on muscle architectural adaptations, I attached the link to two papers, one that discuss the architectural adaptations to eccentric vs. concentric training in vastus lateralis muscle and a very recent one by Timmins et al., which look at the effect of eccentric loading on muscle fascicle length.
Jose L Mauriz
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Melatonin signal transduction in skeletal muscle during  aerobic exercise and Prevention of DNA damage in The elderly?
 
This is a very interesting question. Melatonin has anti-inflammatory effects in different situations (see our review Mauriz et al, 2013). In aging, melatonin has anti-oxidative and anti-apoptotic effects in liver (see our papers Mauriz et al 2007, and Molpeceres et al 2007). Moreover, in exercised aging animals changes in AMK and GLUT have been described by Mendes et al (2013).
Dmitry Kovriguine
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I am researching with a loading suit that not only aims to provide a gravity-like load through the body but also is made from elastic properties. I am trying to quantify in a number of ways the physiological mechanisms by which it "intensifies" exercise. I hypothesised that it may increase muscle activation or something similar, due to the increased load but then I realised that the influence of elastic material stretching and recoiling over a joint has not been taken into account. I think this needs to be quantified before I can understand what to expect in a muscle activation sense. If someone has a skeletal muscle computer model then it may be possible to start to understand how the elastic properties may influence the muscle's work. Thanks!
 
Rudenko, O. V., & Sarvazyan, A. P. (2014). Wave anisotropy of shear viscosity and elasticity. Acoustical Physics, 60(6), 710-718. Rudenko, O. V., Gurbatov, S. N., and I. Yu. Demin. (2014) Absorption of intense regular and noise waves in relaxing media. Acoustical Physics, Volume 60, Issue 5, pp 499-505. Hello, Julia. Drop me a line: are these papers consistent or made just for a report? A good theoretical prototype of skeletal muscles can be done as a stack of flat plates held together elastically with gelatin. Is it true to provide true diagnostic information on muscles? https://www.researchgate.net/profile/O_Rudenko/publications
Lan Wei-LaPierre
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What are the common ways to assess mitochondrial dysfunction in skeletal muscle cells?
 
you can also measure mitochondrial ROS production using a mitochondrial targeted probe, o2 consumption in both isolated mitos and whole cells (in line with the ATP production in PP), mitochondrial Ca2+ uptake during stimulation and mitochondrial enzyme activity using either biochemical assay or histology assay. 
Shahnaz Hasan
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I am interested in the relation between muscle fatigue in the skeletal muscles(eg. mm Quadriceps) and frailty. sincerely,  Edmund Berduszek
 
It is interesting question as you want to see the relationship of fatigue and frailty.You have to measure the MVIC strength of quadriceps muscle and fraily of quadriceps muscle.
Jaakko Hentilä
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Has anyone tried to measure Phospho-eIF2α (Ser51) using the antibody from cell signalling ID ((119A11) Rabbit mAb #3597)) in mice skeletal muscle?
 
We have also measured this with western blot succesfully many times!
Alfonsina Ramundo-Orlando
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I'm wanting to isolate plasma membranes from mouse skeletal muscle to study GLUT4 translocation. Just curious about what an isolated muscle will give me. Downstream applications will be western blotting and radioligand binding. 
 
Dear Derek Bone, we used rat liver about 40 g (from three adult rats) as starting material to obtain about 1.5 ml of plasma membrane suspension (we didn't weight the final pellet) containing an avarage of 10-15 mg/ml of protein (Lowry method). If you need more details please don't hesitate to ask us. Best regards Alfonsina Ramundo-Orlando
Cesare Gargioli
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I am trying to culture the MDSCs from human skeletal muscle and the protocol I'm following is the one published by Eberli et al. in 2009 (http://www.sciencedirect.com/science/article/pii/S1046202308002016). I'm using the same collagenase and dispase digestion, and ending up with a mass of hypercontracted fibres and mostly fibroblasts. Does anyone have any advice with respect to the handling and digestion of the muscle during the isolation so as to ensure myofibre viability? I'd be very grateful for your input!
 
Hi Sujata,  every lab utilizes its own protocol to isolate myogenic precursor, we use collagenase typeII (Gibco) 100U/ml 45 min a 37°C. The human muscle biopsy is kept in Hank's solution at 4°C not more than 48hours. The muscle is finely minced before digestion ( 45min at 37°C) centrifugation, pellet resuspension in aMEM 20%FBS filtered in cell strainer (100, 70 and 40um) and then plating on plastic at low confluence (1000cells/mm2). I hope this will help you, good luck.
Joseph Roche
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Cardiotoxin from Naja mossambica mossambica is commonly injected into skeletal muscle to induce experimental muscle.  It appears that this product was sold exclusively by Sigma-Aldrich.  The product has unfortunately been discontinued.  I would be grateful for advice on suitable alternatives to induce experimental muscle damage in rodent models.  Thank you!
 
Thank you, Dr. Houweling.  Do you happen to know if there are any known differences in the timeline for damage and regeneration for CTX versus notexin? 
Sasha Bogdanovich
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I need the biochemical protocol for skeletal muscle creatine kinase (ck-mb) estimation.
 
Hello Vishnu; First, is it human or animal speciment. If it is animal: There is no standardize protocol for CK measurement, because protocol might be significantly different and they depends from analysis and condition. Protocols depend from kit to kit. You should use the instructions, from the kit you plan to use. Human sample: As mentioned above, any pathology lab can do this easily, and you should check with them from protocol, they might vary as well, or if you have a kit, follow the kit instructions.
Rodrigo Fernandez-Gonzalo
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I am looking for information about any method using microdialysis to assess skeletal muscle protein breakdown. There is a paper (Tesch et al 2008) using microdialysis and assessment of 3-methylhistidine, but this technique may present some limitations. Anyone knows a better one? Looking forward to more information, Thanks!
 
Human models
Lars Larsson
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Hello!  My name is Anna Chen and I am a PhD candidate at the University of Chicago.  I am looking a novel transgenic mice strains and trying to determine if our genetic manipulation is causing fiber type switching.  I am identifying fiber type by frozen section MHC isoform staining.  I was wondering which muscles I should stain and analyze to see the change.  I was going to do tibialis anterior (predominantly fast twitch) and soleus ( predominantly slow twitch) but was wondering if I should try any others.  Thank you very much for your input!
 
Hi Anna, If I were you I would go for soleus and EDL to start with.  These muscles are of similar size.  Personally, I would not determine MyHC isoform changes by immunocytochemistry.  A more precise and reliable method in my experience is to separate MyHC isoforms on SDS-PAGE . Good luck
Jose Sofia Ruffino
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Hello, I am a PhD candidate at the University of Chicago.  I am trying to conduct a western blot on some skeletal muscle tissues isolated from mice.  I am running into problems with the sample prep step.  Normally the tissues are harvested as a whole tissue, immediately frozen with liquid nitrogen, and stored in -80oC.  When I want to do a western I will remove the tissue, suspend it in RIPA buffer that contains protease and phosphatase inhibitors, mince the tissue with scissors and homogenize before I spin the samples down (15 min at 12K in 4 oC) and use the supernatant. According to my Bradford assay I have protein (10-40 ug/uL normally) but when I do a western my internal control ( I've tried GAPDH and Beta Actin) are very very low and my target protein does not exist at all.    Does anyone have suggestions on how to better extract proteins?  Thank you in advance!
 
Hi Anna, try this lysis buffer (ice cold): [20 mM Tris (pH 7.8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 % Triton X-100, 10 % (w/v) glycerol, 1 mM EDTA, 1 mM dithiothreitol] supplemented with protease (Thermo Scientific) and phosphatase inhibitor cocktail (Millipore). We homogenised samples on ice using a dounce homogeniser (40–50 passes) and incubated for 1 h at 4 °C with continuous rotation. Samples were then centrifuged at 13,000 rpm for 5 min and the supernatants were collected. The protein content of the supernatant was determined using a bicinchoninic acid assay (BCA) (Thermo Scientific). How much protein do you load? We loaded 50 µg of each sample. Also, it may be worth checking your Western Blotting technique - have you done this before (perhaps using cultured cells). You can check that the proteins have been transferred efficiently using Co-omassie blue on the gel or Ponceau red. Your housekeeping protein usually stains more greatly than your protein of interest so it may be that you aren't loading enough proteins or that there is an issue with your transfer.  Hope this helps! José
Gordon L Warren
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desmin and vimentin shows upregulation at gene level of skeletal muscle treated with fluoride. please provide me with possible reasons.
 
How is it being treated with fluoride? By intramuscular injection? Was the muscle injured by the treatment? Increased desmin and vimentin expression have been used as markers of regenerating muscle fibers.
Lars Larsson
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They told me that Isopentane is no longer available 
 
I suggest that you use liquid propane cooled by liquid nitrogen instead of isopentane. Propane has a lower freezing temperature than isopentane and you get more rapid freezing with less freezing artifacts.  Further, if I remember correctly, isopentane smells good but it is far from healthy to inhale the fumes from isopentane.  Build a cooler and   freeze your samples in liquid propane
Gommaar D'Hulst
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All the housekeeping genes we use are changing after ischiadicus cut (severe muscle atrophy).
 
Thanks John, much appreciated. But you have to keep in mind that skeletal muscle atrophy is not a straightforward 'injury' proces. The muscle cells stay intact, the just get smaller. Agreed? In fact, we've measured 3 housekeeping genes (RPL4, GAPDH and CYCLO) and they all go up (3-5x!). So you suggest that we do a couple more and analyze them by using GeNorm?
Tausif Alam
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I'd like to measure skeletal muscle glycogen content in mouse muscle samples. Does anyone know and efficient protocol or available commercial kit to measure from small amount of samples?
 
I am not aware of any quick method, as in using a probe or a dipstick!  ;) The old standby method for glycogen assay (Passonneau and Lauderdale, Analytical Biochemistry 60 (1974), 405-412 ) is still a commonly used method. It relies on acid hydrolysis of glycogen into glucose, followed by its conversion into glucose-6-phosphate by hexokinase, which is then converted to 6-phosphogluconic acid by G-6-P dehydrogenase (G-6-PDH) in the presence of NADP leading to a quantitative conversion to NADPH based on available G-6-P. The conversion of NADP to NADPH provides a measure of quantity by spectrophotometer (or in some cases by fluorescence).  If you are not experienced in linked enzyme assays where muiltiple enzymes are employed and certain amount of optimization of proportions and optimization of conditions becomes necessary, you may consider a ready-made kit. There are several companies that sell such kits (not very cheaply), for example: http://www.abcam.com/glycogen-assay-kit-ab65620.html https://www.caymanchem.com/pdfs/700480.pdf There are a few precautions you must follow. Due to endogenous enzymes and cofactors, if the tissue sample is not handled carefully, glycogen could degrade to varying degree, which will create a large error in your measurements. The best way is to snap freeze the the tissue sample in liquid nitrogen, powder it in a mortar pastel (pre-chilled) in LN2 and then resuspend in homoginization buffer if assay is done immediately or store it at -80ºC (or in LN2 freezer) until assay is performed. This aspect of complexity does not change irrespective of whether you mix your own reagents or use a kit. Best...
Wangang Zhang
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In particular, I am interested to know if there are isoforms of troponin for skeletal muscle and laboratory tests to measure them.
 
It is easy to measure the isoform and the amount of tropnin using western-blotting. In skeletal muscles, there are three isoforms including Troponin-I, Troponin-T and Tropmin-C.
Yi Lun
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Hello, I read that the optimal freezing condition would be LN2-cooled isopentane.  The main problem in my case is that we use an outside vivarium which does not supply LN2.  I am wondering whether dry ice-cooled isopentane would work. In addition, if I want to cut cross section, what should I do to make sure that the muscle is at resting length? thanks in advance for any input, Yi
 
Hi Stafano, thank you so much and I will try the recommendations in the link you forwarded. Best, Yi
Rosemary L Sparrow
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This would be of value in determining if blood is a sink for muscle derived reactive species. 
 
Mature RBCs will only survive for a relatively short time in cell culture conditions at 37 oC.  Many will spontaneously hemolyse and release hemoglobin and cell debris into the culture medium, which is not typical of what happens in the healthy body.  You would need to think carefully about the design of your experiment and include the appropriate controls.  
Guillaume Giraud
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Starting from cross-linked minced tissue (pectoralis major, chicken). Thanks in advance!
 
You can check this paper in Methods Mol Biol: Methods Mol Biol. 2012;798:517-30. doi: 10.1007/978-1-61779-343-1_31. Isolation of nuclei from skeletal muscle satellite cells and myofibers for use in chromatin immunoprecipitation assays. Ohkawa Y1, Mallappa C, Vallaster CS, Imbalzano AN. Best.
Cesare Gargioli
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I have read that AP (alkaline phosphatase) identifies pericytes in skeletal muscle (Cossu 2007 and 2011), but are there other ones? Many thanks in advance,
 
Hi Osvaldo, AP is the best one to look at muscle pericytes, I stained muscle section and many of capillaries are labeled. Otherwise other good markes as breedge state are Ng2, CD146 and PDGFRb (CD140b).
Radoje Milić
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Currently working on thesis, which deals with electrolytes/hydration and muscle fatigue in swimmers.
 
Skeletal muscle fatigue is defined as the fall of force or power in response to contractile activity. Both the mechanisms of fatigue and the modes used to elicit it vary tremendously. Conceptual and technological advances allow the examination of fatigue from the level of the single molecule to the intact organism. An dvantage to the study of muscle fatigue is that it is an acute, quantifiable event. In general, fatigue researchers report the magnitude of the fall of force or power in response to a specific contraction protocol designed to produce fatigue. Another approach often used is that of quantifying fatigue as the duration a contraction or series of contractions can be maintained at a specified, submaximal target tension. A review of studies that pursue this challenging area of research follows, and includes a brief description of the tools and techniques used to assay each site in vivo. More on this topic can be found in the relatively recent book Human Muscle Fatigue, edited by Williams and Ratel NewYork: Routledge, 2009.
Philipp Treskes
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I am culturing human skeletal muscle cells on 6 well plates. Initially for the first week they are happy and healthy, then after a week, before I begin to differentiate them, they start to die from the edge of the plate inward, so only a 1.5cm diameter patch of cells remain. The cells don't just die though, it is if they have dried out leaving an imprint on the flask. The cells are in 2ml/well of media and I have tried two different brands of plates. Any suggestions what is happening or how to prevent it?
 
I am amazed. These cells do not look good and certainly do not look like something you would expect from a commercial product under culture with the appropriate commercial medium designed for them. In my hands skeletal muscle isolations always grew enormously well, regardless of the use of coating and most supplements. The cells normally tend to be very robust. I would suggest seeding your cells at high density on collagen-I coated (5 ug/cm2) standard culture plastic dishes (nunc is appropriate here). Fibronectin coating is also possible, but not cost-effective, gelatin generally also works, if you do not have collagen available. Coating the plates, seeding cells at high density and allowing them to reach 90+% confluence might be enough for your cells to recover. Normally their doubling time should be less than 3 days. Your differentiation medium is fairly standard and should work fine. You could also check the pH of your growth medium. Most formulations contain a part of DMEM, which might shift your pH to non-ideal ranges (DMEM was designed for use in 10% CO2 incubators). Always good to check. If you do not succeed in rescuing the cells, you should honestly think about a complaint. I am fairly certain your cells might have been unhealthy from the start, given their phenotype on the presented microphotographs. Let me know how it goes Philipp
Martin Hofmeister
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IL-15 seems to be related to SIRT1 and PPARδ in the skeletal muscle in response to exercise.
 
Quinn LS, Anderson BG, Conner JD, Wolden-Hanson T. IL-15 overexpression promotes endurance, oxidative energy metabolism, and muscle PPARδ, SIRT1, PGC-1α, and PGC-1β expression in male mice. Endocrinology. 2013;154(1):232-45. doi: 10.1210/en.2012-1773. http://press.endocrine.org/doi/pdf/10.1210/en.2012-1773 Quinn LS, Anderson BG, Conner JD, Wolden-Hanson T, Marcell TJ. IL-15 is required for postexercise induction of the pro-oxidative mediators PPARδ and SIRT1 in male mice. Endocrinology. 2014;155(1):143-55. doi: 10.1210/en.2013-1645. https://www.researchgate.net/publication/263162325_IL-15_is_required_for_postexercise_induction_of_the_pro-oxidative_mediators_PPARdelta_and_SIRT1_in_male_mice Molanouri Shamsi M, Hassan ZH, Gharakhanlou R, Quinn LS, Azadmanesh K, Baghersad L, Isanejad A, Mahdavi M. Expression of interleukin-15 and inflammatory cytokines in skeletal muscles of STZ-induced diabetic rats: effect of resistance exercise training. Endocrine. 2014;46(1):60-9. doi: 10.1007/s12020-013-0038-4. Yang HT, Luo LJ, Chen WJ, Zhao L, Tang CS, Qi YF, Zhang J. IL-15 expression increased in response to treadmill running and inhibited endoplasmic reticulum stress in skeletal muscle in rats. Endocrine. 2015 Feb;48(1):152-63. doi: 10.1007/s12020-014-0233-y. Crane JD1, MacNeil LG, Lally JS, Ford RJ, Bujak AL, Brar IK, Kemp BE, Raha S, Steinberg GR, Tarnopolsky MA. Exercise-stimulated interleukin-15 is controlled by AMPK and regulates skin metabolism and aging. Aging Cell. 2015 Apr 22. doi: 10.1111/acel.12341. [Epub ahead of print] http://onlinelibrary.wiley.com/doi/10.1111/acel.12341/epdf
Cleber Ferraresi
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My OD value had a huge variation between each trial, even blank one. The worst thing is blank two has higher OD than the corresponding sample no matter if it was diluted or not. The repeatability of the assay kit is not good. My protocol is as follows.
 
Hi Shengye Zhang, This is the protocol for BCA assay. I used this product from Sigma. It is easy to do and reliable. You just need take out a part of your muscle homogenate and quantify following the instructions.
Fariba Biyouki
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Which filters are the best to use?
 
Dear Dr. Degelaen, I entirely agree with previous posts. To sum up, I am of the conception that a high-quality EMG recording is dependent on skin preparation, electrode type, electrode shape, electrode size, electrode material and construction, electrode placement and fixation, inter-electrode distance, as well as the specifications of the employed electromyograph. In addition, there are multifarious filtering techniques. However, depending on the recording environment, muscles, defined tasks, as well as the interested subsequent signal processing fashions, it is necessary to firstly determine the probable sources of artifacts and noises contaminating EMG recordings and, afterwards, adopting appropriate and functional techniques to diminish their undesired influences as far as possible. Digital Butterworth filters of order 4 have been recommended by some EMG investigators to simply attenuate some high-frequency and low-frequency noises. Regards,
Jeremy P M Mogk
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When reading EMG literature the terms smoothing the signal and RMS sometimes entangled into the same entity and sometimes separated as digital filtering and quantification. Why?
 
RMS can (and has) been used to process EMG in much the same way as applying a linear envelope. By squaring the raw signal, you effectively transform it into a zero-mean signal. Then the mean (and square root) are applied using a moving time window, the length of which the user can define. The selected length of the window (i.e., the time constant) will impact how "smoothed" the signal becomes. The selected time constant will also impact how much the signal gets shifted (similar to the phase shift induced by applying a single-pass linear envelope).
A researcher
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.
 
may be this paper is helpful Direct Isolation, Culture and Transplant of Mouse Skeletal Muscle Derived Endothelial Cells with Angiogenic Potential
Pedro Henrique Imenez Silva
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We are having limiting success detecting NBC1 in human skeletal muscle with Western Blot. Not heating the samples seems to improve blot detection, but we are seeing a band at about 120 kD (predicted is 150 kD). Using Bis-Tris gels seems to work slightly better than Tris-HCl gels. We are currently using self-cast gels, but may try switching to pre-cast gels. Any suggestions would be appreciated.
 
I don't have experience with NBCe1 blots, however, abcam antibody predicts a 130 kda protein, what was also observed by Perry et al (Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells). Perry et al used an antibody provided by Dr Walter Boron (at that time at Yale, now at Case Western). Probably you have reliable results. EDIT: Is your blot clear enough to be quantified?
Chandra Mohan
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It has recently been shown that the earliest defect in insulin resistance occurs in skeletal muscle in the form of inhibition of glycogen synthesis. Is there evidence of glycogen toxicity in skeletal muscle, or why cant the muscle increase its capacity to store glycogen like adipose tissue does with fatty acids?
 
90% of our glycogen is stored in the liver and 10% in muscle. Each gram of glycogen comes with 4 grams of water. Hence, we can not store too much in muscle. Also muscle lacks glucose-6-phosphatase., Hence, muscle glycogen breakdown does not produce glucose, but makes lactate. Too much muslce glycogen breakdown will produce excess amount of lactic acid.
Daniel A. Boullosa
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Back in 2003, WADA banned gene doping. But is it even possible with current technology? If gene doping was possible then one would expect that all monogenetic disorders would be cured by now. Is it something that WADA should be concerned about into the future? http://bbc.in/L8WLn4
 
Good point Alex. Let me comment however that overexpression would be an advantage to one or few factors (not a complete physiological function) while top-level performance is the result of the non-linear interaction (complexity) of lots of factors. For example, one sprinter could improve his maximum strength with genetic doping but what about the risk of tendon rupture?
A researcher
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I have to detect myofibers degeneration/necrosis on muscle sections. I know evans blue is the most used method, however, it has to be administered in live animals and I have muscle sections. I decide to detect IgG, is it a right method? Does anyone knows something else?
 
dear Valeria recent paper detect IGF-I as indicator for muscle degeneration please check this paper http://www.sciencedirect.com/science/article/pii/S006512811300161X
Sara Benedetti
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Seeking to learn more on the matter.
 
I completely agree with previous answer, for growing muscle cell lines DMEM with 10% FBS, 1%PS and 1%glutammin whereas for differentiation DMEM 2%Horse serum plus 1% PS and 1% glutammin always work with any cell type with spontaneous myogenic capacity. In case you are thinking to isolate/culture satellite/myoblast cells from muscle (so primary cells) or to culture human muscle cells maybe other media (richer media) will be more suitable.
Joseph Gosnell
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Tetranectin has been suggested to play a role in tissue remodeling, due to its ability to stimulate plasminogen activation and its expression in developing tissues such as developing bone and muscle. I would like to study its role in muscle damage due to exercise, and if there is some relationship between them.
 
Here are a few articles I found on the subject. The last article (Tetranectin is a Novel Marker for Myogenesis during Embryonic Development, Muscle Regeneration, and Muscle Cell differentiation in vitro) is probably the closest to what you are looking for. Hope this helps and best of luck in your research. 
Johnathan Nuttall
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I was wondering if anyone looked at AMPK activation (phosphorylation of T172) in-vivo in skeletal muscle or heart after overnight fasting. I would expect it would be activated, but I could not find any evidence in the literature. Any advice would be greatly appreciated.
 
I doubt it because I don't think the glycogen in skeletal muscle is depleted during sleep unless. The liver glycogen store supplies glucose to the circulation. However, muscle doesn't have the glucose-6-phosphatase that would be required for glycogen to be converted to glucose and leave the cell. In other words the glycogen stored in your muscles is only depleted when your muscles need the energy. If you went running your muscle AMP kinase will become activated, but I'm doubting that an overnight fast would do it.
Salim Ansari
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FISH with Y chromosome-specific probes can be done, but it is time-consuming and relatively expensive.
 
logically, We can use barr body as marker to discriminate between male and female nuclei. histone macroH2A1 staining is used to identify barr body due to histone macroH2A1 content of histone protein which play important role in silencing of whole X chromosome. Best regards.
Lael L Cheung
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Our research group currently has some goat primary antibodies and we wish to use these to examine human skeletal muscle on western blot. Our current hrp conjugated rabbit anti goat secondary is giving us not specific binding at about 45kDa which is the same size as a protein we are interested in. So I was wondering has anybody found a good hrp conjugated anti goat secondary which they have used on human skeletal muscle samples and does not give not specific binding? We have tried a few different blocking steps and varying secondary antibody concentration but its not really suitable.
 
Perhaps the authors of the following publication can offer some meaningful suggestions (see section on "Western blot analysis and antibodies"): http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3707125/
Anita Quigley
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I have been doing some cryosectioning on frozen human skeletal muscle samples. The initial samples I worked with were not embedded in oct and were fine to cut on the cryostat. However, I now am working on some different human skeletal muscle samples which had been covered in oct and then frozen down via isopentane cooled liquid nitrogen. These oct covered samples are not proving easy to section. In fact, when the blade comes into contact with the muscle tissue itself, it is not cutting through the muscle nicely. Instead, it seems as if the human skeletal muscle tissue which has been covered in the oct is much tougher than the muscle tissue which was not covered in oct. I want to use the oct covered tissue for immunohistochemistry. Before the sectioning of the samples, I remove the samples from a -80°C freezer and put them into a cryostat at -22°C. I typically leave the samples from 30min - 1hr before I start the sectioning in order to allow the samples to de-freeze somewhat from the previous storage temp of -80°C. However, this protocol still results in the oct samples being difficult/impossible to section satisfactorily. I was therefore wondering if anybody had encoutered a similar problem, and if so, how was it resolved? If anybody has any other suggestions please share them as they may benefit others who may are experiencing something similar.
 
Hi Peter, We freeze our human muscle in liquid nitrogen cooled isopentane. Never liquid nitrogen..the freeze damage is too extensive in our experience. We actually cool the isopentane till its just starting to freeze over on the bottom before we drop the muscle in. It's important to get good quality isopentane with low water content...And dont reuse the isopentane too much. Colleagues of ours actually mount the muscle on a small strip of card, as human muscle can be quite soft (depending on what muscle is biopsied and from whom it's from, age, lipid content etc etc) and plunge the sample into the cooled isopentane by holding the card with tweezers. That way the muscle is always orientated correctly and doesn't deform in an undesirable manner on freezing. I havnt done this myself, but it seems to work well for them. I dont have too much trouble just freezing the muscle by itself but It might be somthing to try if you are having issues. Just before freezing, carefully blot moisture from the muscle with some fine grade tissue to prevent ice crystal formation on the surface of the muscle then immediately freeze. We leave the sample in there for 10 to 20 seconds before removing to a cryovial in dry ice. As Clare mentioned, agitation by swirling the isopentane gently seems to help. remove sample with isopentane cooled tweezers. Cryovials are best for long term storage rather than standard laboratory tubes..so specimens don't dry out in the freezer -80oC. Separate tweezers for handling frozen and unfrozen muscle too is always handy! For sectioning, we mount on the chuck with minimal OCT. the chamber and chuck are cooled between -20oC to -25oC. We cut sections between 5 to 8 microns. Use a fine brush to remove any ice crystal formation on the blade (from breath). We mount on super frost plus slides.
James Imre Nagy
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There are several sub-types of pannexin and connexin channels that are expressed either during embryogenesis or in different tissues in adults. From your experience, does anybody knows which ones are expressed specifically in skeletal muscle in vivo or in muscle cell lines (L6, C2C12)?
 
Cx39 is expressed in skeletal muscle, low in adult but at higher levels in early postnatal periods. These work was done by the group of Klaus Willecke. Search Pub Med and Cx39. Antibodies that work and which we have tested are available from Life Technologies (Invitrogen).
David Sassoon
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Given some of the recent papers from Karyn Esser's lab using the satellite cell depleted mice, are satellite cells still worth studying or should we divert our attention to gross muscle biology like protein synthesis changes?
 
Not to tout our own work, but we published a recent review on this and related issues: see Trends Mol Med. 2012 Oct;18(10):599-606. doi: 10.1016/j.molmed.2012.07.004. Epub 2012 Aug 8. Stem cells in the hood: the skeletal muscle niche. Pannérec A, Marazzi G, Sassoon D. We also have a manuscript under revision to be re-submitted very soon on this issue. It would appear that satellite cells are not really necessary for myostatin-block induced hypertrophy as a few others have reported, HOWEVER, it is important to note that the genetic models in mice in which satellite cells are depleted initially result in a marked decrease in fiber size. There is also evidence that an initial short term hypertrophy of fibers is satellite cell independent whereas maintenance of this hypertrophy may require satellite cells. We also see a crucial interaction for the myogenic population of PICs that are not satellite cells and do not express wither Pax3 nor Pax7 unless recruited into the myogenic lineage. My oversimplified view is that satellite cells are not the whole story and that other cells in the muscle tissue are centrally involved in 'growing' the muscle and giving it plasticity. This would make sense if you consider that exercise induced hypertrophy also involves increased vascularization, increased connective tissue and other adaptive tissues changes that support the fibers and their satellite cells.
Jakub Ziak
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Does anyone have a standardized protocol for isolation of skeletal muscle mitochondria from rodents?
 
Hello, we are using (sligtly adapted) protocol developed by Rasmussen et al. - http://www.ncbi.nlm.nih.gov/pubmed/9324953. Instead of bacterial proteases we are working with trypsin (as trypsin together with collagenase is used for primary muscle cells isolation) - 2,5 % trypsin 3x diluted in ATP medium.
Mohammad Ahmad Braikeet
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skeletal muscle relexant ,GABA agonist
 
Swiss albino mice (SWR)Swiss albino mice (SWR)
Eric K Patterson
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I dont know if ordinary tap water will work, I assume it will cause the capillary bed to collapse. I am also looking at the negative effect of exposing live cells to tap water. The kicking off of coagulation may lead to blockages as well, has anyone ever tried this experiment?
 
Well there's nothing that's going to be "the same" as blood, but I've been involved with experiments where we used RPMI 1640 + 2% BSA to perfuse lungs/heart for several hours. The mice had heparin injected directly into the heart before the perfusion began to stop coagulation, and then the blood in the lungs/heart was flushed with the RPMI. I don't think anyone in our group ever tried, but I assume, that water would not perfuse for very long.
Revathy Carnagarin
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ELISA for Pi3k in rat skeletal muscle
 
Echelon Pi3K assay kit has been mentioned in several publications.
A researcher
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So far no commercial kit has worked. Has anyone successfully done this? I know it is difficult using skeletal muscle and not cultured myoblasts.
 
You might isolate nuclei and then extract the RNA for example with Trizol, this will be more difficult with sarcoplasm; may this is helpful: http://www.springer.com/cda/content/document/cda_downloaddocument/9781588299772-c3.pdf?SGWID=0-0-45-593709-p173800903
Paul T Reidy
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We know that FSR plateaus at around 20g of whey (1.1 g leucine) in human skeletal muscle, but does anyone know this dose response for mTOR signaling?
 
Gommar, Interesting question.  Unfortunately, it likely does have a clear answer from in vivo studies in humans. Here is a nice set of studies looking at manipulations of AA and Leucine in human muscle. http://www.ncbi.nlm.nih.gov/pubmed/24284442 http://www.ncbi.nlm.nih.gov/pubmed/22451437 Here are a few more studies (that have muscle protein synthesis and mTORC1 signaling) looking leucine and or AA dosing in humans.  http://www.ncbi.nlm.nih.gov/pubmed/20844186 http://www.ncbi.nlm.nih.gov/pubmed/15596483 The paper you may be looking for is here http://www.ncbi.nlm.nih.gov/pubmed/25107987 The answer to your question depends on a variety of factors, including, but not limited to, age, health status, physical activity level, presence of other nutrients among others. Supposedly aging, inflammation or underlying health issues may necessitate a need for higher levels to stimulate mTORC1 signaling and protein synthesis.  As a person become more physically active the sensitivity to amino acids is thought to improve and the amount of AA/leucine needed is likley lower. An important point to remember is that more mTORC1 signaling may not actually mean more muscle protein synthesis. See http://www.ncbi.nlm.nih.gov/pubmed/20844186   At a certain point, the signal may just act an an on/off switch.  In such a case of over stimulation, if the stimulus is too high, a negative feedback loop could start to desensitize mTORC1 signaling. as discussed here http://www.ncbi.nlm.nih.gov/pubmed/11498541 If you want the answer in cell culture or animals I don't have as many resources, but you could start to look at these. http://www.ncbi.nlm.nih.gov/pubmed/24646332 http://www.ncbi.nlm.nih.gov/pubmed/24806638 http://www.ncbi.nlm.nih.gov/pubmed/21702994 http://www.ncbi.nlm.nih.gov/pubmed/10947949 http://www.ncbi.nlm.nih.gov/pubmed/19882215
Christian Q. Scheckhuber
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We posit that, by virtue of its location in the ER/SR and Golgi networks in skeletal muscle, and the finding that some dysferlin mutations are associated with ER stress, dysferlin is likely to be involved in protein processing and trafficking.
 
Some studies indicates that dysferlin may bind lipids in a calcium-dependent manner, consistent with a role for dysferlin in regulating fusion of repair vesicles with the sarcolemma during membrane repair ( please see this article: http://www.ncbi.nlm.nih.gov/pubmed/24461013 ). However, how dysferlin achieves a 're-closure' of the membrane is not clear yet... Best regards!
Ioannis Kokkinopoulos
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Fibro/adipogenic progenitors are most often identified by removing CD45 and CD31, and labeling CD34 and Sca1. These progenitors are important regulators of skeletal muscle regeneration and are therefore relevant to a number of muscle degenerative diseases, aging, and cachexia. However, to me, their relevance seems deminished because Sca1 is not expressed in humans, nor any other vertebrate besides mouse. Why do scientists then so commonly use it when other markers, ie PDGFR are available?
 
Although an equivalent Sca1 marker in humans is elusive, by using this model, it is possible to a) find an ortholog of it b) understand the mechanisms behind these cells, which could lead closer to a therapeutic approach, once a similar marker/cell type is identified in humans.
Ugo Carraro
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Hi everyone. I would like to show that the skeletal muscle of the animals that I am studying have a high number of necrotic fibers. Any of you knows if there is any specific measurement, apart of CK activity in plasma? Thank you in advance
 
You my look for troponin leakage in the serum, but the standard is to i.p. inject in vivo Evans’ Blue dye (EBD), collect, frozen, crysection and observe by immunoflourescence the permealized muscle fibers AND the MyHC-emb- positive myofibers that are direct evidence of muscle fiber necrosis occurred during the last then days before muscle harvesting. GOOD WORK AND RESULTS ...
Lara Araujo
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Elderly rural black ethnicity
 
Olá, Professor Luiz. Creio que esta pergunta não tenha ainda um ponto de corte bem definido para a população que o senhor está estudando. Em nosso serviço, com idosos com idade acima de 80 anos, temos usado os pontos de corte indicados na referência McLean et al. J Gerontol A Biol Sci Med Sci 2014 May;69(5):576–583. Talvez, para idosos mais jovens e com maior atividade física os pontos de corte sugeridos pelo consenso europeu de sarcopenia ou considerados na referência Alley et al, J Gerontol A Biol Sci Med Sci. 2014 May;69(5):559-66. Possivelmente, o senhor ajudará a responder à esta pergunta se puder caracterizar sua população na capacidade funcional relatada por questionários (Kats, Pfeffer, ou Lawton), na auto-percepção de saúde e no desempenho físico (com handgrip, teste de sentar-levantar, caminhada em 400m, velocidade de marcha em 4m, equilíbrio) e fizer as correlações entre handgrip e velocidade de marcha, entre outras. Um abraço, Lara
Pablo M Peixoto
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I would appreciate feedback (and protocols) on whether or not it is feasible to establish functional synapses between motor neuron and skeletal muscle cell lines. Thank you! P.
 
Thank you!
Claude Lachance
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The study of Coffey et al. (2009) compares the effect of diverse contractile activity in human skeletal muscle. They measured phosphorylation of several proteins and the results (phospho/total) are log-transformed. I don't understand the interest of these method.
 
Hi Frederick, one reason to log-transform the data is that upregulated or downregulated data (compared to a control sample) will not be normally distributed, which can be problematic if you want to use a parametric statistical test (t-test or ANOVA for example). The reason is that upregulated data can have values from 1 up to infinity while downregulated data will have values from 1 to 0 on a linear scale (4 times upregulation has a value of 4 while 4 time downregulation has a value of 0.25). By log transforming the data, upregulated and downregulated expression data can then have a similar scale of changes. Hope that make sense
James R Bagley
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Hi! Im studying adipose and skeletal muscle tissue atm. As a quick sanity check of Cuffdiff results on isoforms detection and quantification, I want to look at well described isoforms. What I mean is to look at i.e. two isoforms that occur in a fixed ratio within the tissue. And then to see if the RNAseq results also quantifies the same ratio. Does anyone know of such examples for skeletal muscle/adipose tissue, or have other sanity check methods (both dry- and wet-lab based). Thank you! I also asked a similar question here: http://seqanswers.com/forums/showthread.php?p=150370#post150370
 
Hello Sindre, I'm not sure if I can answer your question directly, but these papers may provide some useful information (or point you in the right direction). There are several “references genes” (i.e. housekeeping genes) in skeletal muscle that are (supposedly) universally expressed. Perhaps you could take the ratio between two of them? http://www.ncbi.nlm.nih.gov/pubmed/20045408 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0001854 http://www.biomedcentral.com/1471-2199/9/79 http://ajpregu.physiology.org/content/294/1/R192 I hope this helps, James
Martyn Alun Sharpe
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I would like to use Mitosox red o read mitochondrial ROS. I have seen that mostly it is used for imaging purposes. How reliable it is to use it in 96 wells plate assay mode? If somebody has standardized the protocol is it possible to share the reference? I will be using it for C2C12 skeletal muscle cells.
 
Rashmi , I understand that cell death would change the number of cells. However, if I have determined cell survival for that particular cause "X", then I know how much it contributes towards it. Can you please explain that argument again ?? Thanks !!" Here is the problem. You have mitochondrial signal and a small background signal from the proteins sitting at the bottom of the plate. Each control cell gives you a signall of 100%, background give 5% and you have 70% of the plate surface area covered with cells. Now you add 'X' to half the cells and at time t add MitoSox. Now anything that is going to increase mitochondrial superoxide generation is going to be cytotoxic. The more ROS generated, the more cell death. Imagine if treated cells give a signal of 600%, but dead cells give 0%. Small changes in cell numbers convert a signal of 600% into lesss than background. You get titrations that begin at 100%, rise and then fall. The ROS levels rise, but total cell numbers fall. You cannot use Hochst or other viability dyes to count cells and normalize, as dead/dying cells tend to be brighter.
Christian Q. Scheckhuber
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Can anyone suggest a method for isolating mitochondria from skeletal muscle cells and/or electrocytes?
 
Hello Heather, in the attached article you will information on how to isolate mitochondria from skeletal muscle cells. Best regards!
Annunziata Nancy Crupi
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Please, does anyone have a detailed protocol for isolating macrophages from rat skeletal muscle? The Most protocols I found peritoneal macrophages and adipose tissue. I need these cells remain viable for culture of M1 and M2 after injury.
 
I usually omogenize skeletal muscle tissue as I do to extract mononuclear cells (using collagenase and dispase), then I use a FACS-sorting to isolate macrophages, selecting for a specific antigen (e.g. mac-3 or F4/80). I use mice, not rats, so I think you will find a higher amount of macrophages than me. 
Stephen Andrew Bustin
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After treating isolated RNA to cDNA for the first time in mice skeletal muscle I got 8-11 in 15 samples and 1 value at 16. In previous studies, my colleagues I've worked with were getting values that are a bit higher.
 
Although it is of course correct to say that the Cq reflects the cDNA input into your qPCR reaction, the cDNA input is dependent on the amount of RNA you have reverse transcribed in the first place. Hence, if you start off with  the same amount of RNA between samples, your Cqs will be a reflection of the original RNA concentration. However, the efficiency of conversion from RNA to cDNA is RT , sample and target dependent, and can vary considerably (see Stahlberg's two papers in Clin Chem. In 2094). But assuming you are using the same amount of RNA  from each sample to start off with and the same RT you can quantify your RNA with reasonable accuracy, and if you are using relative quantification this san be within 5-10 fold, depending on how much RNA you are analysing.
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I started some work on skeletal muscle repair and regeneration recently. Due to lack of experience, I'm confused at choosing the cell surface marker to identify regenerated muscle cells in rats for IF and IHC staining. Could anyone give me some advice?
 
Hi hong What is the model for regeneration induction ? if you want to detect muscle regeneration you can stain with either neonatal myosin or embryonic myosin These markers expressed by nascent myotubes as well as the regenerated one Best regards 
Matheus P S Magalhães-Gomes
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I am using immunohistochemistry on 10um cross section of tissue to identify cross-sectional area of muscle fibers obtained from a biopsy sample. Images attached
 
I am currently looking for an automated method as well, but till now I can't find an easy and reliable method. I measure CSA from gylcolmethacrylate (Leica Historesin) sections 5um cut and stained with toluidine blue. I use the AxioVision Software from Zeiss which I manually measure each muscle fiber. I also would like to find another method to save my time.
Eric A F Herbst
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Are there any articles that point out under what conditions/circumstances not to use alpha-tubulin as a loading control? Is it ok to use alpha tubulin as the control in skeletal muscle?  What a good/better control for muscle?
 
alpha tubulin is an excellent loading control in skeletal muscle. Never had or heard of problems with it in any circumstance.
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See above
 
Use chilled buffer (50 mM TrisHCl, pH 7.4 containing MgCl2, 250 - 270 mM sucrose and protease inhibitors).  Place sample in chilled Petri dish and chop with sterile surgical blade.  It would be nice if you have a homogenizer, just be careful about heating or increase in temperature. all the best regards Arshad 
Revathy Carnagarin
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I am planning to examine the effect of a new protein on insulin dependent phosphorylation events up to GLUT4 translocation. I am designing my entire study on this and plan to use C2C12 but I wanted to be very sure before I start.
 
Thank you very much Anton. just starting with work and will see to that it is differentiated into myotubes. I have started with C2C12 and HSMM.
Osvaldo Contreras
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I would like to perform a double immunostaining in muscle tissues. The antibody of my interest is Rabbit anti-Protein X (PX). Moreover, the mouse antibody anti-Protein does work properly. I have plans to correlate the localization and expression of the cells expressing PX with Fibronectin in muscle tissue. So, I would appreciate your help. Thanks! :)
 
Terry Partridge
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I mean in a wild type condition (adult) and without damage. I am looking for some publications in this field. Thanks.
 
The answer to this question depends on the level of sophistication behind the question. Besides the various types of muscle fibre. There are satellite cells, which are thought to be the main source of myogenic cells for repair of damaged muscle fibres.  There is some evidence of heterogeneity of satellite cells - some being more stem-like than others.  Skeletal muscle is richly vascularized and thee is some debate about the interactions of some of the microvascular cells and satellite cells. There is also some evidence that pericyte cells of the microvasculature can become myogenic.  Recently, an interstitial cell call fibroadipocyes have been shown to interact with myogenic cells during muscle repair.   In addtion to all of the 'resident' cells, lymphocytes and cells of the mononuclear series have been shown to pass through even 'normal' muscle, but it should be remembered that normal muscle is in constant interaction with the environment via the stresses and strains of normal physiological use.
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In some patients using the antihypertensive "Co-tareg 160/12.5" and "Isoptin 240 retard" as a co-drug, they complain of chronic fatigue and perhaps minor stiffness in some skeletal muscle, particularly the neck and shoulder muscles.
 
Yes, it has been reported. Parkinsonism is a frequent side effect of some calcium channel blockers (CCB), especially cinnarizine and flunarizine. CCB-induced parkinsonism (CCBIP) usually improves spontaneously after discontinuation of the offending drug, but many patients exhibit persistent symptoms. See also: Calcium channel blocker-induced parkinsonism: clinical features and comparisons with Parkinson’s disease Pedro J. Garcı´a-Ruiza, Feelix Javier Jimenez-Jimenezb,*, Justo Garcı´a de Ye´benesa. But L-type calcium channel blockers of the dihydropyridine class may have potentially neuroprotective effect and protect again Parkinson's disease - "L-Type Calcium Channel blockers and Parkinson’s Disease in Denmark" Beate Ritz, MD, PhD,1,* Shannon L. Rhodes, PhD,1 Lei Qian, PhD,2 Eva Schernhammer, MD, DrPH,3 Jorgen Olsen, MD,4 and Soren Friis, MD4 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2917467/
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Increase in diet hardness? Atrophy of the contralateral masseter muscle by botox injection? I would appreciate any advice you may have.
 
have a look Tanne K. Current status of temporomandibular joint disorders and the therapeutic system derived from a series of biomechanical , histological , and biochemical studies. APOS Trends Orthod. 2015;5(January):4–21.
Mehdi Hedayati
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Is there any specific protocol to quantify vitamin C concentration in skeletal muscle tissue samples? While scanning the literature I came to understand the HPLC assay is the standard method to measure vitamin C; however, is it feasible to quantify Vitamin C by HPLC from skeletal muscle too? Thanks in advance for your responses.
 
Dear Annadurai, Hello, you can determine Vitamin C content of muscle homogenate superannuate based on reaction between Vit C and PTR (Phosphotangestate Reagent) in 1:1 V/V ratio. Absorbance in 700nm must follow. you can see attached paper for details. http://www.znaturforsch.com/ac/v57c/s57c1062.pdf
Andrew D'Lugos
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Staining 10um cross sections with PAS and immunofluorescent (for fiber type, cell membrane). Need assistance with imaging/ analysis.
 
Thank you Gundrun, I am including an amlyase control stain along with my PAS stain. I am having a hard time figuring a way to measure the optical density of each fiber.
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I know there are a number of protocols and techniques for studying mitochondrial dysfunction and apoptosis such as TUNEL assays, immunoblots and so on. Can we use PCR for this purpose? What markers should be considered? I am especially interested in doing this for rat skeletal muscle.
 
What are you looking for specifically? With rat skeletal muscle, muscle atrophy or exercise usually moves total mitochondrial content and function (we examine max respiration as a typical measure) in either direction in unison. That evidence is collectively out there. Typically we determine mitochondrial respiratory function in rat muscle through respiration experiments in isolated mitochondria or permeabilized fibres (to get kinetics). The kinetics can change independent of mitochondrial content in muscle, but if you're looking at just PCR, you can probably only use this as a marker of content, particularly if you're interested in apoptosis / mitophagy. We have a paper coming out shortly examining how changes in mito content in muscle vs other tissues occur with exercise , I can keep you posted on that, but I think Joe Quadrilatero's lab does a lot of mitophagy/apoptosis work which may be more applicable?. Mitos have other functions too, however, such as Ca2+ sequestration, ROS generation, etc., so the definition of "mitochondrial function" is a little ambiguous? I'm used to referring to ox phos capacity. Of course with PCR there's always mRNA expression of certain genes encoded by the mitochondria as well, but not sure how this is effected by apoptosis. There may be other ways to infer mito dysfunction from PCR, but I've yet to come across them / am unaware of them. Hope this helps?
Adam Lightfoot
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I found 2 studies in which it was found to be working well in skeletal muscle of mice http://jap.physiology.org/content/112/11/1839 http://journals.plos.org/plosone/article? Sincerely, Jaakko
 
I've no experience with the kit you mention. However, we've used the Cell Biolabs OxiSelect™ Protein Carbonyl Immunoblot Kit in both murine and human muscle tissue in our lab to great success.