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Hello,
I have no prior experience with growing cell line nor differentiation toward specific cell type. Currently I have C2C12 cell line which I need to grow and then differentiate it towards skeletal muscle cells.
Can you please provide me with as much as possible information, SOP, tips and tricks?
Thank you
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Hi Aleksandar Dishkelov I am using high glucose DMEM.
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Hi everyone, I am looking for a human skeletal muscle cell line which however requires differentiation times of 7-10 days and which can grow in horse serum. Could someone recommend me an easily manageable line?
Thank you a lot
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I am working on skeletal muscle synthesis, and I did Western to see p70s6k.
I expected I could see the p70s6k band around 70 kDa, but I saw two unexpected bands below (attached).
I am not sure how to interpret the bands.
Western information that I did is the following:
10% gel__Running 50v for 30min and then 100v for 2h__Transfer membrane (PVDF) for overnight in cold room (36v)__Cell signaling Primary (rabbit)
I would appreciate it if you leave any comment on that.
Sincerely,
Sonny.
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Thank you so much for your reply.
Sonny.
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We have infected mice with an AAV-EGFP and we were hoping to directly visualize the fluorescent signal in frozen muscle sections without antibody staining.
Any suggestions would be greatly appreciated.
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Dropping muscles directly into liquid nitrogen causes severe freezing artifacts -- ice crystals will form, which causes crushing/tearing of the muscle fibers. To avoid these artifacts, muscles can be frozen in isopentane cooled in liquid nitrogen: place a small container of isopentane in a larger container of liquid nitrogen; wait until the isopentane starts to freeze; you are ready to put in your muscle.
Good luck!
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Dear sir,
I am going through your paper titled " Micromechanical modelling of skeletal muscles based on the finite element method " . You have mentioned force velocity characteristics with equation (numer 9 and plotted the graph (figure 4)).In that relation kc , ke are the curvature and d is the offset of the eccentric function.
My question is kc,ke , d same for all kind of muscles ? if not how to get the values of kc,ke , d values for different muscles?
Could you please reply me sir.
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To answer your question, the values of kc, ke, and d in the force-velocity relationship may not be the same for all types of muscles. The force-velocity relationship is influenced by several factors, including the muscle fiber type, the size and length of the muscle, and the contractile history of the muscle. Therefore, the parameters kc, ke, and d may vary depending on the specific muscle being modeled.
In order to determine the values of kc, ke, and d for different muscles, experimental data is typically needed. These parameters can be determined by fitting the force-velocity relationship to experimental data obtained from measurements of muscle force and shortening velocity under different conditions, such as varying levels of activation or different types of contractions. Alternatively, these parameters can be estimated using a combination of experimental and modeling techniques, such as inverse modeling or parameter optimization.
It is also important to note that the force-velocity relationship is often simplified in muscle models, and may not fully capture the complex interactions that occur within the muscle during contraction. Therefore, while the parameters kc, ke, and d can provide useful insights into the behavior of skeletal muscles, their values should be interpreted with caution and should be validated against experimental data whenever possible.
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We are trying to isolate nuclei from mouse skeletal muscle but at the end of the process, the clumping of nuclei was still present in the slide. We were not getting good-quality of nuclei for library preparation and further genomics study.
As a resuspended buffer, I used RNAse inhibitor, 2% BSA and 1X PBS buffer. So, I would like to know how to minimize/remove these clumping nuclei and get better nuclei quality at the final stage after the resuspension process. Please anyone can help me how to solve this problem?
Thanks in advance
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Traditionally, Non-ionic detergent is used for isolating nuclei from tissues. It is the detergent that causes nuclear clamping. You can try detergent-free method or a detergent -containing kit but is able to generating single nuclei.
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Skeletal muscles are stained with the blue color but other half of the picture was stained with red color. Can someone tell me what kind of tissue or cell is it?
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It seems that the fixation and also the cutting procedures had some problems. Moreover, my feeling is that you may had some problems in freezing the tissue, as it seems that some cells were de-freezed.
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I'm looking for a protocol for measure SDH activity in skeletal muscle homogenate. Are there any protocols that do not include NaCN or KCN?
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Georgia State University Alumni Association's 40 Under 40 Class of 2023 (gsu.edu)
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working on analyzing skeletal muscle cells exit form myogenic differentiation.
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It will depend on the species studied. For exemple, in chicken cells slow MyHC are expressed and sarcomeric myosin (MF20 antibody) will be also used.
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During examination of transmission electron microscopy images of skeletal muscle biopsies from patients suffering from different myopathies, sometimes I find somewhat unusual mitochondria with white spots. Initially, I assumed that is some sort of oxidative stress damage or even artifact, yet now I think I could be wrong; usually, in patients with frequent "white spotted" mitochondria, I find more pronounced pathological changes in mitochondria (such as paracrystalline inclusion or onion-like cristae) in other fibers, sooner or later.
That made me think, so I've tried to find similar images in other studies, but to no avail. I have no reasonable explanation as for now. Any ideas?
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Dear Rabeah Al-Temaimi, thank you for your response!
Images shown in the mentioned article are somewhat similar to ours, it's quite possible that our mitochondria may also lose their membrane potential. Early or late apoptotic events are also possible, as I find in muscles with severely disorganized myofibrils.
Cristae swelling is rather unlikely (yet still possible) - on image taken on higher magnification (attached below) I cannot see swollen cristae, those hyperintensities seem to be independent of visible cristae location. Specimens for current study were fixed in 2.5% glutaraldehyde right after biopsy - however, I am not present during tissue collection, it's possible that between biopsy and muscle fixation more time passed.
Alpers-Huttenlocher syndrome can be rather excluded - it has early onset and we have adult patients only - although, from what I see, muscle mitochondria looks similar indeed.
Thank you again for your response, it was very helpful! Now I have some foothold for further research.
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Hi fellow researchers, has anyone ever worked with AICAR to induce activation of AMPK in L6 skeletal muscle cells? So far I tried to activate it by adding AICAR (either 0.1mM or 1mM) in DMEM with 1% pen strep to L6 myotubes. This was done after serum starvation for about 24hours. However, the AMPK was not activated when I compared with the untreated group. Has anyone ever activated it in these types of cells?
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Jeffrey J Brault Thank you for your response and recommendations. We'll optimize the time of treatment following these and others in literature through a time point study.
Interesting, I'll read more on how serum starvation increases pAMPK and whether the effects are additive. This wasn't my question but will definitely look into this too.
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I would like to understand how different load on the muscle affects the development of bone tissue, the presence of tubercles on the bones, thickening, etc.
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Attached is a review article we wrote several years back about the linkage of muscle loading to bone properties and how that changes with aging.
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I'm currently working with Western Blotting technique for human skeletal muscle. However, when i use the Cell Signaling antibody (#8164 and #14179) no image were reveled. Apparently, i dont have problems with WB protocol, once others antibody brands (e.g., Santa Cruz and ABCAM) works for others proteins. In addition, the lysate protocol and the WB protocol used were performed according Cell Signaling recomendations.
PFK (D4B2 - 8164S ) - Rabbit mAb - Lot: 5
HIF-1alpha (D2U3T - 14179S) - Rabbit mAb - Lot 4
Has anyone had similar problems with Cell Signaling antibody? If yes, how to solve this problem?
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Check if your AB is ok by SDS PAGE (Coomassie staining) and/or Western Blot (probe the blot with anti-species HRP).
If it is ok, do you have a positive control for PFK?
If in doubt, include another positive control for your detection system, i.e. an AB or serum from rabbit in this case.
I would not blame brands in general, esp. not CST. Have you called their tech service for assistance?
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Dear colleagues,
I was wondering whether there are any ways or any softwares that I can use to analysis the muscle cross sectional area in my H&E histology images?
I tried to use ImageJ thresholding, unfortunately it does not work efficiently for me. Thus, I was wondering whether there are any currently established methods.
Thank you very much in advance.
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I also want to calculate muscle fibers CSA in H&E staining using imageJ. Thresholding cannot be used but manually it can be done and it will be time taking process. I want to know about how many and how the fibers are randomly selected in a sample field, how many sample fields will be needed and what should be the magnification?
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I am working on natural and synthetic insulin mimetics and insulin potentiating agents. I am assessing the effect compounds on glucose uptake in L6 myotubes, for that I want use Insulin as positive control. However I am doubtful that human insulin can work on cell lines derived from rat.
Any clarifications is appreciated. Thanks in advance
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Thank you Yasuhiro Nishida I will check for it
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For the past few weeks we've been using three different cell types on the Fura 2 calcium experiment including iPSC's, RenCells, and human skeletal muscle cells. We loaded our cells with 2.5 uM of Fura 2 in HBSS + 1% BSA. We incubated the cells in both 37 degrees or at RT for about 30 minutes and hydrolyzed for about 20 minutes in HBSS. Using the Olympus microscope and MetaFluor software we were able to see the cells with the fluorescent microscope indicating the loading was successful, but saw no response when exposed to the drugs we used. As a positive control test we used 5mM glutamate or 20uM ionomycin and also saw no response. I appreciate if anyone could give any insight on potential issues or solutions we can take to successfully run this experiment.
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Did you load the cells using the acetoxymethylester form of Fura-2? Your comments suggest you did. Use fresh DMSO to solubilize the Fura-2 AM. Which excitation filter set are you using? Which emission filter? Also ensure your cells are in glass and not plastic chambers. Plastic absorbs UV wavelengths more so than glass.
If you can't get Fura-2 to work, switch, at least temporarily, to using Fluo-3 or Fluo-4. They are easier to work with.
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I used proteinase K as a protein digestion step before using a Qiagen AllPrep DNA/RNA extraction kit. I have used it before on WT murine heart and skeletal muscle tissues and got good yield. Today, when I used it on my experimental samples, I got virtually no DNA or RNA.
I added the appropriate amount of ProK to my samples and incubated for 10 mins at 55 degrees as recommended. Then, I spun them down and left the samples on ice while I ate lunch (~45 minutes). This break was the only difference in my protocol today versus my first test run.
I was hoping that icing my samples would inactivate ProK, but I believe that it remained active and digested all of my DNA and RNA.
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Proteinase K should not ever digest DNA or RNA, unless it is contaminated with nucleases. Make sure you use a DNase- and RNase-free source of the enzyme.
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what dye I can use to look for Ca2+ distribution in muscle cell?
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If you want to study calcium dynamics in skeletal muscle fibers, many calcium sensitive dyes works but the selection depends on many factors including: Imaging equipment available (Framerate, UV and/or Visible excitation); necessity of real calcium concentration (ratiometric vs single wavelength dyes); which type of calcium event (quiescent, stimulated or small events like sparks) and even the location (cytosol, sarcoplasmic reticulum or mitochondria).
Without knowing your specific need and equipment, is difficult to recommend something. However, For general use:
I had successfully used in isolated skeletal muscle fibers and whole EDL muscle ratiometric UV dyes (Indo-1, Fura-2) and singlewave visible dyes (Fluo-5F and Rhod-2) for stimulated calcium events (single twitch or 40 Hz tetanic).
Fluo-4 is good for smaller calcium events, but almost too bright/sensitive for stimulated events.
Fluo-5N can be used for study calcium in sarcoplasmic reticulum.
Hope that helps!
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Hello, i did H&E staining using cryosection.
However, there are so many holes in myofiber.
Here is my protocol.
No fixation for myofiber staining
1. Dissection - skeletal muscles
2. Embed into O.C.T using liquid nitrogen
3. After completely frozen, stored at -80c before using
4. Put it into -20c cryostats 20 mins before sectioning.
5. Cut to 10μm thickness
However, it is really hard to cut without getting holes, even though the blade is brand new.
Is the problem of water crystalization or freezing time?
Please help me.
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The holes seem to have formed due to ice crystals rupturing the tissue. If the experimental animal wasn't perfused with saline followed by PFA, I would recommend a post-fixation of the tissue.
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Hello, everyone. I would like to determine the fiber type of each skeletal muscle fiber based on its SDH and ATPase activity.I used the dry ice-hexane method to freeze freshly isolated skeletal muscle because the liquid nitrogen freezing method produced bubble artifacts. Although these methods were used to evaluate by enzyme activity, there is unavoidable bubble artifact.
If it is difficult, I will prepare frozen sections after saturation with normal sucrose and use an antibody-based staining method for each muscle fiber.
If you have any knowledge, I would appreciate it very much.
Thank you in advance.
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The most common procedure to avoid artifacts is to freeze muscle in isopentane cooled in liquid nitrogen. In other words, the isopentane is added to a small container, and this container is put in a larger container containing liquid nitrogen. When the isopentane is cooled to its freezing temperature -- it starts to becomes a white solid -- then it is ready.
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I was wondering if there is a reason to use one method over the other. Some literature used acetone and ethanol (3:1), while others used acetone and methanol (1:1). Which mixture do you think leads to better results? And why?
I'm staining for skeletal muscle proteins (e.g. MHCI, COXIV, PLIN proteins ...).
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I assume you are talking about fixation for light microscopy and not both light and electron microscopy. If the former, why fix at all? Why not cut frozen sections from unfixed muscle in a cryostat? If a staining procedure requires fixation (many don't), you can do it during the staining procedure and the fixation procedure can be done custom to the staining procedure.
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i don't get any DNA a the end of the protocol, i tried BEB buffer modification too but nothing works, any clue?
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The above may be helpful. Trizol is for RNA purification, if it does not work, you could try another anothermethod. good luck.
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I am looking to submit a brief review for publication. Brief reviews are similar to mini reviews in their requirements. However, I am looking to see which journals best fit the scope of my paper. It's related to aging, cardiovascular health and skeletal muscle performance. The only journal that I have found so far that says they accept brief reviews that also has submission guidelines for a brief review is: International Journal of Sports Physiology and Performance. I would like to see what other options I might have beyond this journal. Any help would be appreciated. Thank you.
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Here are some posibillities:
Journal of Exercise Physiology - but there are charges for submission and publication
- they do not state what is the length required for a review
Journal of Clinical Exercise Physiology
- for review/commentary type articles, you need to write to them (at the journal) first to discuss your topic before they will consider your idea for an article
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I am woking with type II diabetes and doing assay on L6 skeletal muscle cell line to understand the glucose uptake by using on-cell western method. But I am confused Do i need to protein quantification for On-cell western?
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As per the protocol, protein estimation is needed. I don't run western blots but run a coomassie protein assay to determine protein levels of the samples against standards. In our lab, we have never used western quantification as it is not needed.
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Could someone help explain to me why have researchers mostly focus on the GSP muscles ( gastrocnemius, soleus and plantaris) , but not the tibialis anterior (TA) in the hindlimb suspension model?
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Ta Hu I use the Aurora system as well to study both dorsi and plantar flexion. We do the study in vivo using the foot plate so we can assess it weekly. If you want gastrocnemius specific force then you will have to do a non survival surgery where you cut off the tendon and attach solely the gastrocnemius to the lever arm. That will provide more information such as optimal length. When I use the foot plate i don't measure solely gastrocnemius, since it is plantar flexion,, both gastroc and soleus are involved.
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Hi everyone,
I am working on a grant on muscle Na+,K+-ATPase activity in human subjects. However, our lab does not have a protocol on the assessment of Na+,K+-ATPase activity in skeletal muscle homogenates. Although I could find some info in relevant papers, it is still hard for me to have a precise idea about the procedure and the reagents/devices needed. Could you let me know where I can find a step-by-step protocol?
Thank you in advance!
Shelly
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My guess would be that the kit might not have a specific protocol for preparation of muscle samples... and the inhibitors in the kit might not be specific enough for the other ATPases in skeletal muscle. This kit was used quite recently to study activity in skeletal muscle (Journal of Physiology), so it might be worth looking into:
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Ø Before starting the muscle biopsy procedure, pour 100 mL (approximately 1/3 of beaker) isopentane (2-methybutane) to a glass beaker or a steel beaker.
Ø Suspend the beaker in a bath of liquid nitrogen (either in a larger beaker or Styrofoam box) and leave until the isopentane ice crystals start to form on the bottom of the beaker and on the sides of the beaker. When this occurs, isopentane has reached optimal freezing temperature (-150 °C). Time taken averages between 3-5 minutes.
Ø Following the skeletal muscle biopsy, remove the excess moisture by dabbing the tissue on the filter paper. Place muscle in proper orientation. Cover it in OCT (Optimum Cutting Temperature compound), and lay it flat on a labeled disposable freezing mold. Freeze the muscle for about 10-12 seconds (Being very small muscle tissue)
Ø Transfer the frozen tissue on to dry ice and let the isopentane evaporate( approx. 20minutes) and wrap the tissues in aluminum foil and store them temperature (-70 °C to -80 °C) .
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I have done this a few thousand times. Your procedure should work. I might do a couple things differently. 100 ml of isopentane is more than I would use. It will take a while for the isopentane to cool down. However, if you are willing to wait, you can use 100 ml. The most important step, I believe, to getting artifact-free sections is to cool the muscle specimen/OCT as quickly as possible. I will plunge the specimen/OCT to the bottom of the cooled isopentane as fast as possible. I do not let the specimen/OCT stay in the isopentane more than 10-12 seconds. If it stays longer, I often see cracking of the OCT.
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I recently carried out ELISA on L6 skeletal muscle cell lysates using Elabscience GLUT 4 Elisa kit in order to determine the GLUT 4 expression in the cells when treated with different treatments. However, I have been getting no expression in all my treatments including the positive controls although, my standards for the Elisa produced a good curve. I tried to use different methods to extract the cell lysates and i confirmed that the treatments were done properly but there isn't any difference when I repeated the ELISA. All the results from the ELISA appear negative. May I know if anyone has used ELISA before to determine GLUT 4 expression in these cells? Which extraction method was used? or perhaps is there another better method to determine the expression?
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Javier Russell-Guzmán Thank you for your response. Yes I am also doing western blotting for the GLUT 4 as I am mainly looking to determine its quantity in my samples after treatment. The part about the GLUT 4 translocation is also really insightful and helpful, I will read more about it. How about the ELISA though? Is it not recommended to use for determining the GLUT 4 quantity in skeletal cells?
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perhaps the same mechanism governs lateral force transmission in a single skeletal muscle cell? (Sybil Street in J Cell Physiol and Science)
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Sten Knutsen's latency relaxation of the thin filaments
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I used this kit for measuring Total TG content in rat Plantaris muscle. However, I have some questions about the calculation of the final TG concentration.
It is highly appreciated if anyone can help me.
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Thanks, Gordon for your reply.
Actually, I want to make sure that my calculation is correct.
Briefly, the final concentrations were normalized based on muscle weight and then multiplied by 2, because I separated extracted lipids in two vials equally. This is how I calculated lipid concentration.
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I am looking for a non-NMR based (preferably) method of analyzing glycogen synthesis in the skeletal muscle and heart. Can anyone share a protocol where one has used radiolabeled glucose incorporation into muscle glycogen in vivo?
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Dan Robbins Thank you for your reply. Yes, I am aware of that paper that you mentioned. But I am really looking for more of a "real-time" method of glycogen synthesis.
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Usually skeletal muscle after injury undergo regeneration process following by all key phases of degeneration, inflammation and so on so until regeneration of fibers. For example we usually identify regenerating myofibers on sections by centrally located nuclei, however I am curious about how long (time/days) it take further for the regenerating myofibers to be mature, when the centrally located nuclei will already be moved to periphery of membrane as fibers 'll be mature.
Any valuable suggestions would be greatly appreciated, please.
Thank you..!
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These journal articles should address what you have requested:
Laumonier T, Jacques Menetrey J. Muscle injuries and strategies for improving their repair. J Exp Orthop. 2016;3:15. doi: 10.1186/s40634-016-0051-7.
Karalaki M, et al. Muscle regeneration: Cellular and molecular events. In Vivo. 2009 (Sept);23(5):779-796.
L. Baoge, E. Van Den Steen, S. Rimbaut, et al. Treatment of skeletal muscle injury: A review, Int Scholarly Res Notices. 2012; doi: 10.5402/2012/689012.
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Spinal Muscular Atrophy is inherited disorder. Defective gene is the SMN1 gene which codes for SMN protein. Defect in this gene or its absence can cause death of motor neuron in the spinal cord leading to weakness of voluntary muscles.
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Spinal muscular atrophy is inherited in an autosomal recessive pattern
There 4 known types
Spinal muscular atrophy type 0 is evident before birth and is the rarest and most severe form of the condition. Affected infants move less in the womb, and as a result they are often born with joint deformities (contractures). They have extremely weak muscle tone (hypotonia) at birth. Their respiratory muscles are very weak and they often do not survive past infancy due to respiratory failure. Some infants with spinal muscular atrophy type 0 also have heart defects that are present from birth (congenital).
Spinal muscular atrophy type I (also called Werdnig-Hoffmann disease) is the most common form of the condition.
Spinal muscular atrophy type II (also called Dubowitz disease) is characterized by muscle weakness that develops in children between ages 6 and 12 months. Children with this type can sit without support, although they may need help getting to a seated position.
Spinal muscular atrophy type III (also called Kugelberg-Welander disease) typically causes muscle weakness after early childhood. Individuals with this condition can stand and walk unaided, but over time, walking and climbing stairs may become increasingly difficult. Many affected individuals require wheelchair assistance later in life. People with spinal muscular atrophy type III typically have a normal life expectancy.
Spinal muscular atrophy type IV is rare and often begins in early adulthood. Affected individuals usually experience mild to moderate muscle weakness, tremors, and mild breathing problems. People with spinal muscular atrophy type IV have a normal life expectancy.
In autosomal recessive inheritance, a person who has SMA has inherited two altered (mutated) copies of the SMN1 gene from his or her parents. The parents of an individual with an autosomal recessive inherited disorder such as SMA are carriers of one copy of the altered gene
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We are trying to probe for the RpS6 protein which typically shows up around 32 kDa on a western blot. We have two questions regarding probing for this protein:
1. We recently ran the protocol for soleus muscle, and the protein showed up around 15 kDa. What could be the cause of this? Could it be that the protein broke down before we probed for it, or that this is the result of nonspecific binding? Any speculation is welcome.
2. When we probed for the protein in another muscle (gastrocnemius), no band showed up at all. RpS6 is a ribosomal protein, so we figured it would be present in this muscle and that there was an error in probing for it. We are using PVDF. Thanks.
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RPS6 protein has phosphorylation sites, and its phosphorylated at S235 & S236.
1. The showing up of 15kDa in western blot might be due to loss of phosphorylation sites due to denaturing and reducing condition of SDS PAGE in western blot. Probably your antibody is detecting the non-phosphorylated form only.
2. When probed for another protein, no band might be due to reason that your antibody is recognizing non-phosphorylated form and there is only phosphorylated protein RPS6 in muscle.
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I am trying to measure differences in eNOS activity in skeletal muscle in sedentary and exercised mice, following the conversion of 3H-arg to 3H-cit, and assaying the samples with and with out L-NAME to get eNOS activity. However, I am having multiple problems, from signal no different from the blank (even with ~300 ug protein, fresh 1.25 mM L-NAME, fresh buffer components (including FAD, FMN, BH4, NADPH, CaM) and plenty of CaCl2), to no difference in activity with and without L-NAME, to huge variance between replicates (my pipetting is not that bad!!). I have 10 uM Arg total (1 uM 3H-Arg) in the assay, and allowing the samples to react for 30 min at 37C. I stop the reaction with NaAcetate pH 5.5 / 5 mM EDTA, and then pass the solution over dowex AG 50W x8 (Na form) to bind unreacted Arg in the solution. I am happy to share my protocol with anyone who can help me figure out what I am doing wrong!! Thanks for any and all help!
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Hi Adele,
You may try to remove myoglobin from your skeletal muscle extracts, which tends to interfere with many enzymatic reactions. My 2 cents...
Gedas
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I have purchased the Horse skeletal muscle Myoglobin (M0630) and Sodium dithionite (157953) from Sigma Aldrich. During my experiment, Mb (concentration 11 μM to 17 μM) gave its absorbance at 409nm in UV-Vis spectrophotometer but after adding the 100μl of 100 μM Sodium dithionite, the shift in the soret maximum from 409 nm to 435nm was not observed. Rather, the intensity of the Mb peak was reduced. An even higher concentration of sodium dithionite gave further reduction only in the soret band of Mb obtained in the 409 nm. The buffer I used to dissolve the components was 0.1M Phosphate buffer (pH-7.0) and incubation time was 5 to 30 min.  
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Thank you Gordon L Warren.
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I have mostly been using the immortalised C2C12 murine cell line for in vitro skeletal muscle atrophy studies. In my lab, I used serum-free DMEM for 24 hours to stimulate muscle atrophy in C2C12 myotubes, which was validated via upregulation of Atrogin-1 and MurF1 through RT-qPCR. I'm planning on using the human immortalised myoblast cell line LHCN-M2 for validation. Reading the protocol I found serum-free media is required for differentiation. So my question is what methods can I use to mimic atrophy in LHCN-M2 myotubes?
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Muscle cell cultures are already in an atrophy state, i.e., there is minimal (if any) contractile loading. Perhaps it would be better to go in the other direction, i.e., increase contractile loading, so as to promote "hypertrophy". Electrically stimulate the cell cultures. This is harder than it sounds. Too much stimulation can kill cells.
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I will be freeze-drying small skeletal muscle samples (~60mg) per sample using a Virtis SP Scientific Benchtop Pro Freeze Dryer. I've had difficulty findings a reference protocol to determine the optimal length and would love input. There is seemingly plenty amongst various animal models using larger samples (200-300mg) running them for ~24h, but I'd like to save time if possible.
Has anyone seen any such methodology and with the above device if possible?
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Hello Steven,
Key in this kind of drying it a path out of the tisssue for the water. Muscle or other animal tissues can be a complex mix of substances, and it is therefore challenging to model the drying process well. Typically, dry until you reach a constant mass is the method, however, how you weigh the sample in the dryer is a challenge. This leads to long, perhaps over long, methods for drying tissues.
Challenge here is to keep the water all frozen. The tissues is complex, but also the places that the water exists are complex too, full of peptides, sugars proteins and salts, all of which change the freezing behaviour of water. The low pressure, 200mTorr = -40°C freezing / sublimation point, is a funtion of making sure that the whole sample remains frozen so that there is minimal tissue damage and creation of artefact in your sample. Remember, the vacuum pressure in the system will control the sublimation temperature of the water.
The thing on your side is that your samples are low mass. I would, if you can, ensure they are thin, and prefreeze them. However, the challenge is that most tissues processors use a shelf based freeze dryer which enables the tissue to be frozen on the shelf, this gives far superior control to a benchtop basic lab dryer. It also allows the addition of heat, which when in steady contition speeds up drying to a certain extent. To speed things up in a Benchtop, the best we can suggest is put the dryer in a sunny window such that IR from the sun will help out.
If you can, I suggest a couple of experiments with samples that you don't need for your research, e.g. use some meat from the store, and see how that goes. If you prep this like your samples, load up 3 or 4 flasks and then you can remove them sequentially say at 6 or 8 hour intervals to evaluate the process of drying. With such small samples, counterintuitive though this may sound, it may help the process to pre-freeze them a samll block of buffer to overcome the antifreeze effects of plasma.
Kind regards
Rob
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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.
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Lara Ivanković why don't you try to analyze the samples with an additional method for verification of the results? Inhibition of 1,2,3-trihydroxybenzene is often used ( . Some comparisons with standard kits have also been published ( eg. https://faseb.onlinelibrary.wiley.com/doi/abs/10.1096/fasebj.21.6.a814)
Kits are often ungrateful as, in reality, each analytical method should be optimized for a specific purpose and quality parameters should be satisfied for given conditions (eg. not all kits work with all buffers, with the same sample preparation procedures, ...)
If you decide to double-check with the pyrogallol method I'd be happy to help.
Best,
Jan
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I am running an oxyblot on homogenized skeletal muscle tissue lysate. When I added all the reagents from the millipore oxyblot kit, the samples turned yellow and I got a grainy white precipitate in the mixture. This happened in samples with the DNPH mixture and the control mixture. Is this a normal reaction and if not, any recommendations to prevent this?
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I am not sure about this specific commercial kit, but for the original colorimetric DNPH method, the precipitation is normal and desirable after we add trichloroacetic acid to both test and blank samples, right after the derivatization with DNPH.
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Dear all,
I am looking for reliable antibodies for the adrenergic receptors alpha1D and alpha2A, to use in Western Blotting on human skeletal muscle tissue. Anyone with experience with good antibodies from similar tissue preferably from humans?
I have come across many different antibodies, all of which has since been removed from the supplier, and I am therefore looking for some good experiences or references.
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I recommend monoclonal antibodies from Abcam, Roche, Biocompare, and Sigma. They work excellent.
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Hi all,
I´m stimulating skeletal muscle cultures prepared from human donors. After one week of differentiation I use a C-Pace system with the following conditions: 2-5 Volts, 2 ms, 1 Hz. I use 12 wells culture plates. Although the electrical conditions are not extreme compared to the available literature (for the same commercial system), my cells are damaged by the stimulation. Even only 2 hours of stimulation reduce the number of cells and induce vacuoles and vesicles (visible with transmitted light). I don´t know if the type of culture plate (previous reports use 6 or 24 wells plates) could explain the deleterious effect of the stimulation.
Any experience/input would be appreciated.
Thanks in advance
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Thanks for your advice
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I am looking to quantify both human serum and skeletal muscle homogenate betaine concentrations via ELISA or colorimetric assay kits. Has anyone had a good experience with any products accomplishing this?
The assay we were planning to use recently became discontinued without further plans to restock. Any help is greatly appreciated!
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I am planing to apply EMS on upper extremity muscles and observe muscle using sEMG and MMG. I am wondering how to do the normalization in case of EMS? is it same as in the case of normal sEMG and MMG i.e. dividing the sub-maximal signals with the signals obtained during MVC?
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Hi Jawad, I've always felt it was ideal to at least stick with the same analysis method for both the submaximal and maximal contractions. So if the submaximal stimulation is a series of spikes at a certain frequency, and peak-to-peak (P-P) is the most appropriate method to get the amplitude, then you'd want the same for the maximal signal. However, P-P does not work well with MVCs, so a maximal M-wave from electrical nerve stimulation would be optimal. If that is not an option, and normalization can only occur with an MVC, then I'd use RMS as your analysis method for both the muscle stimulation and MVC (and not P-P).
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We are trying to prepare organoids from myoblasts. Could you help us about the typr of additional stromal cells or any useful methods to built them?
Thanks!
ross
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Hello, did you successfully culture skeletal muscle organoids ?
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creatine digestion, absorption and circulation. How?
Is C digested? Or taken in as a whole?
How is C absorbed by the enterocyt? Are there transporters?
How is C leaving the enterocyt? Sometimes structures get altered in there.
How is C travelling through the blood circulation?
Does C go to the liver?
How much C gets into the circulation comparred to ingestion?
Think about C and skeletal muscle there is enough information.
Thanks a lot!
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Hi Peter,
That is what I always asked myself. And here is where I found a plausible answer
Regards
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Hi Researchers,
We use the trizol/chloroform phase separation extraction method and have been consistently ending up with RNA samples with high (3-5) 260/280 ratios. I have not been able to find much literature for troubleshooting this as most contamination issues/solutions are for low ratios and shifted peaks. It only seems to happen with skeletal muscle and I frequently end up with white, very sticky pellets that need to be manually removed from the side of the tube with a sterile tip during ethanol washes. Does anyone have any suggestions?
I will attach the protocols but in brief: we make our own RNAsol, homogenise tissue in 500uL using hand-held pestles and continue with the chloroform/isopropranol phase-separation extraction method. As skeletal muscle is a fibrous material, I added a 10min 4 degree spin at 12000g prior to chloroform addition to pellet ECM etc. but most recently, for EDL muscle, this did not make a difference. RNA samples generally generate good peaks (low left shoulder, peak at 260) but higher than ideal ratios. We are concerned this causes issues with qPCR. Our cleanup protocol always produces RNA samples with ratios in the recommended range but this makes extraction more time-consuming and lower yield. Using a kit has also addressed this issue but that is expensive and I worry about using this method for smaller tissues weights (ie. EDL vs TA) We do not understand what the contaminant is...
Thanks for your help!
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I did RNA isolation from skeletal muscles with double TRIzol method or combination of Trizol/ RNEasy column and got very good quality RNA. I believe that you might be taking too much tissue and too little RNAzol. Try to increase TRIzol volume (at least to 1ml) and decrease amount of tissue. I collected tissue (appr 20-30mg, or 5mmx5mmx5mm piece) at room temperature- (it's protected from RNAses in Trizol- no need to keep it at LN2 or 4C before final processing), placed it immediately into half of final volume of Trizol (0.5ml), dissected it with scissors into small pieces directly in Trizol, then homogenized directly in Trizol later in a day after collection of all the samples. RNA is protected in Trizol at least 24h at RT. After homogenization added another half of TRIzol (final volume of Trizol is minimum 9 volumes of tissue size but never less than 1mL)- divided TRIzol volumes in half just to avoid spill during homogenization. Then followed regular Trizol protocol. Your protocol looks OK except need of addition of EQUAL to aqueos phase volume of isopropanol. I also usually do 2 washes with 70% ethanol to get rid of salts before air drying of RNA pellet. After resuspension in water and DNAse I treatment I do either Trizol, either RNeasy column RNA purification again. If you don't plan DNAse I treatment (exon spanning primers and verified -RT controls), single round of RNA purification is enough. Once I had larger than normal piece of tissue and this caused exactly the same problem as you described.
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I am performing macrophage staining in the oral mucosa using an IFA protocol. There are oval-shaped structures on my scans that have background stain/autoflorescence. After doing some research, I found they are either cross-sections of skeletal muscle or adipose tissue. They sometimes appear ordered and clumped like they are skeletal muscle cross-sections, while other times they are more sporadic as if they would be adipose tissue. I am having difficulty identifying which is which. Do both tissue types usually autofloresce? If so, how can I differentiate between them while looking at my scans? Thank you!
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hi paper,
you may coupling your macrophage staining with anti myosin heavy chain or perilipin immunostaining to mark fibers or adipocytes, respectively. Alternatively, you can perform a hematoxylin and eosin stain. In this case, adipocytes appear as white vacuolar unstained cells while fibers will be stained in red
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I need to perform live/dead stain on fresh skeletal muscle slide but I am struggling to cut the fresh skeletal muscle to desire thickness.
The plan is to embed the fresh skeletal muscle in 4% agarose. I tried to dry it as much as I could with Kimwipe; also tried to dip it in petri dish of agarose gel, move around then move it to the moulding base (aim is to remove as much water as possible). Unfortunately, the muscles still tend to fall apart during vibratome sectioning. I am not sure if it's due to the residual liquids from muscle or it's just difficult to cut due to the epimysium / perimysium.
I am just wondering if anyone has any experience / tips on cutting fresh skeletal muscle with vibratome, and what thickness you managed to achieve. Thank you.
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Did you consider the approach followed in this article ?
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Dear Researchers,
We have generated an inducible satellite cell-specific knockout model (iIpax7-cre). Our aim is to confirm a negative impact of our KO on muscle development. To do so, we are inducing KO at ~8wo and attempting to injure muscle and induce expansion of the target protein-depleted satellite cells using cardiotoxin injection.
Our lab is new to this and we are observing less effect on muscle development markers in our controls following cardiotoxin injection than we would expect. We are aware of significant batch variation issues in the literature/through collaborators. We have read about other muscle injury models.
Aside from this, I am unsure whether this is the best method to address our aim. Could any researchers with experience in the area please provide suggestions?
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Cardiotoxin works but is not my preferred way for inducing a traumatic skeletal injury. About 20 years ago, we compared it against using a freeze injury. The freeze injury was more consistent in the amount of injury induced. The problem with using cardiotoxin seemed to be related to the volume of cardiotoxin injected into the muscle. In our case, we were using the mouse tibialis anterior muscle. When injection volume exceeded a few microliters, there would be leakage of cardiotoxin from the muscle. FYI, I am attaching an article that describes our freeze injury technique. We do use it for studying muscle regeneration.
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I knocked out a gene which is expressed in skeletal muscle, one is hsp40 and another one is a co-chaperone of hsp90a1. Previous study showed that hsp90a1 is specially expressed in skeletal muscle, which involved in sarcomere protein assembly.
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If you haven't already, the first thing you should do is confirm successful knock out of your gene/s. If a specific antibody is available, confirm the absence of protein by western blot or, even better, confirm its absence in skeletal muscle using IHC. Can you detect evidence of nonsense-mediated decay by in situ hybridisation?
If the knock out is successful, then you can explore potential reasons for the lack of phenotype, where one might be expected. One possibility is compensation by paralogous genes with similar function. Can you detect an increase in mRNA transcripts which might be compensating? Are they expressed in muscle?
Another possibility is that you are missing a phenotype. How are you looking for evidence of poor sarcomere assembly? You should, for example, stain for a range of myofibrillar proteins (e.g. MyHC, actin, alpha-actinin, titin.... etc)...
Cheers,
Mike.
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I am using QIAamp Mini Kit for DNA extraction and having relatively low yields from heart, skeletal muscle and adipose tissue (around 2 ug). I tried to increase the amount of proteinase K and to extend the incubation time at 56 deg C to get complete tissue digestion but it didn't help much. Any suggestions?
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Jana Tumova Thanks Jana!
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A patient with hereditary desminopathy (Thr341Pro DES mutation in a heterozygous state) was recommended to refuse toothpaste. He continued to brush his teeth twice a day with a toothbrush with only water. As a result, within one month we noted a significant increase in strength and muscle mass in this patient. The patient did not take any medications during this period. After 30 days, the muscle condition returned to its original level. How can this positive effect be explained?
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The toothpaste may affect gut microbiota balance of the digestive tract thus affecting natural PH levels. The triclosan is proving s extremely aggressive. https://stm.sciencemag.org/content/10/443/eaan4116
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I am working on C2C12 skeletal muscle cell line in 6 well plate. I want to do Transmission electron microscopy (TEM) of my sample with cell line . how much sample need or where is possible in India.
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What is the size of the structures, etc. you wish to assess? Are these small intracellular structures (e.g., calcium release channels) or ones that cross multiple cells (e.g., components of the extracellular matrix)? How heterogeneous are these structures, etc.? If they are very homogeneous from one cell to the next, then you can get by with a "small" specimen. On the other hand, if the structures are heterogeneous, your specimen will need to be "large" in order to provide a reliable assessment.
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Hi! I am doing qRT-PCR of GLUT4 in mice liver and skeletal muscles tissue in normal condition. The data showed the similar expression of GLUT4 in both tissue. Any one can explain that in which condition GLUT4 is expressed in Liver or in both tissue? Thanks
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Thanks@Valeria Tananska
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Staining 10um cross sections with PAS and immunofluorescent (for fiber type, cell membrane). Need assistance with imaging/ analysis.
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Hi Andrew,
It's many years since you posted this question, but I'm having similar difficulties at present and wonder if/how you resolved this?
Best,
David
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We have tried more typical housekeeping genes (GAPDH, HPRT1, b-actin), but these seem to quite variable. Has anyone had luck with any other housekeeping genes in this tissue? Thanks!
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Hi,
I am working on developing an MRI based imaging method for measuring/detecting creatine recovery rate in creatine kinase (CK) reaction post plantar flexion exercise.
It's 2D variant, with single slice coverage of 10-20 mm thickness, has already been developed. Whereas I am developing it's 3D varaint with
with multi slice coverage (4-5 mm thick slices), while still keeping the same temporal resolution of 30 seconds. So, using my method I can probe multiple slice. This allow us sufficient resolution in plane (~1mm) and also along the muscle length (4-5 mm resolution with ~30 mm coverage).
Since only 3D method can probe the intramuscular heterogeneity in CK kinetic along the muscle length, I am very interested in knowing some skeletal muscle disorders where the assessment of intramuscular heterogeneity along the muscle length may be valuable. This will make my work, allowing multi-slice coverage, more significant. I would very much appreciate some inputs.
One of such disorders could be Duchenne muscular dystrophy.
Regards,
Dushyant
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Dear Prof. Andersson, Apologies for the delay in responding. Somehow I did notice any notification for your reply from research gate. Previously, our lab has developed a method to measure recovery kinetic of creatine following plantor flexion exercise. That method could only image 2D slice and hence, could distinguish inter-muscular difference between say lateral gastroc, medial gastroc etc from a single inplane image. I extended the method to enable mapping along the muscular length i.e. my method is 3D. So, I can measure difference between various muscle groups as well as if there is an abnormality within same muscle. I am writing "introduction" of my paper and would like to demonstrate the significance of my work in various creatine kinease disorders, which are non systemic and hence, the ability to scan multiple slice may bring unique information.
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I have been considering to use either lentiviral or piggybac with Tet-On 3G promoter to overexpress MyoD1 in iPSC lines. I am pretty new in this stem cell research and I came to know from my former colleague that these iPSC lines are difficult to transfect and through electroporation she managed to transfect 30% with simple GFP marker of cells but it was nearly 10-15% when she tried with CRISPR vector. Does anyone enlighten me with my query that which system would be better to go for. Piggybac is simple but it depends on transfection whereas lenti should be more effective with integrating since cells will not need transfection but infection. But virus infection is how much effective for iPSCs??
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Dear Yulia Panina,
Thank you for your reply and I am sorry to write you back late. Could you please elaborate a bit in what perspectives piggybac was better than lenti? I cam to know through One of my colleagues that someone used sleeping beauty method which is similar to piggybac and was not happy with it. Thanks in advance.
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I used B-actin on my gastrocnemius sample but it doens't seem to work. The ponceau stain on my membrane showed pretty equal bands, but B-actin on the membranes showed very unequal bands. Any recommendation on a loading control good for skeletal muscle tissue?
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Best practice (also enforced by many journals including JBC) is to first identify a protein that remains unchanged under your experimental conditions in your hands. If you choose a HKP from literature, you need to still prove stability of the protein under control and experiential conditions. Them after identifying the combined linear range for your target and HKP perform normalization expt.
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Hi everyone
I am working on a 7 segment model of the human body to simulate a walking gait cycle (3 segments for each leg and 1 segment for upper limb).
I have a problem with this model. The order of knee and ankle joints follows the reported orders in standard data (less than 150 N.m in each one of the joints). But the order of Hip joint in each leg is much more than what I have expected. This torque value is about 600-700 N.m.
I wanted to know has anyone faced with the same problem and how should to resolve it?
Regards.
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Hi Akbar
Your results are highly dependent on the degrees of freedom definition. For instance, if you don't personnlize the axis of rotation of the knee, you may have unaccurate values. More particularly for moment which are computed with forces lever arms. How do you define this axis of rotation? Is it constant ?
And, if you wish to better understand your results you should have a look to the decomposition of the joint moment, more particularly for hips (abduction-adduction, flexion-extension and internal-external rotation moments). Or you could compute joint energies and powers.
Even if walking movement is mainly included into the saggital plane, some interesting information may be measured in the other ones.
And, depending on your objective you may also define the upper limbs. More particularly if at last, you would like to estimate muscle force production.
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We have a clinical setting, so getting a biopsy of the heart isn't always easy. we are interested in understanding mitochondrial function in the heart in HF. Can I perhaps use blood cells to measure mitochondrial parameters in the heart? Can I use something else instead?
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Dear you can do that by relating to lymphocytes ratio.
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Hello everyone.
I'm using the NucleoSpin TriPrep kit from MN to extract RNA, DNA and proteins from human skeletal muscle biopsies.
Problem is, I usually obtain very low A260/230 ratio (<1.0 and sometimes <0.5), especially when the quantity of RNA or DNA I'm getting from the biopsy is very small (less than 20 ng total).
I tried ethanol purification with sodium acetate and glycogen but I keep getting very low A260/230 ratio, even though I'm being careful with the washing and dry steps. Worse, I'm losing 30% of my RNA/DNA each time I'm purifying them.
I don't have that problem with larger RNA quantities.
Is it my RNA / DNA solution that is still contaminated after one or even two purification, or is it the spectrophotometer that is not sensitive enough for small quantities of nucleic acids and that is giving me wrong results?
Should I use another protocol to improve their quality?
Thank you very much!
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You might try using a sample clean-up kit like the Zymo Research DNA Clean & Concentrator Kit (https://bit.ly/2HfWwpX), or similar kits from other vendors.We use these often in our Lab.
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Dear all,
I want to perform a KI67 staining on 3D tissue-engineered skeletal muscle of approximately 2cm long and 3mm thick (cilindrical shape). Does anyone already performed a 3D staining with KI67 on tissue with comparable dimensions and if so can I have some tips or working protocol?
I would combine the KI67 staining with a clearing as well (this clearing has already been established in the lab).
Thanking you in advance
Kind regards
Lisanne
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Mouse skin is considerably thinner than a 3-mm diameter cylinder of muscle. Mouse skin thickness is in the 0.5-0.8 mm range.
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I have tried the Ogilvie protocol with prestain buffer at pH 4.3 and 4.5 but the fibers are either all light blue or dark blue respectively. I've come across many papers which claim to use this protocol with mouse tissue and stain all 4 fiber-types, but I don't have any other colors but blue. Am I dehydrating for too long? Can anyone help? I'm interested in changes in the ratios of type II fiber subtypes between experimental conditions. Thanks!
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Sure, I can send you the protocol.
About the sensitivity, I can not 100% guarantee that one reviewer won't ask for ATPase staining... However, I think it will be more likely that someone will ask for a myosin separation gel. At least, this is what happened to us, some years ago.
Moreover, I stayed many years in Stefano Schiaffino's lab, and I can tell you that we NEVER performed a single ATPase staining!
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I am trying to isolate RNA from mouse skeletal muscle but I am not able to get a good 260/230 reading. I am using Qiagen RNeasy kit. I would appreciate any suggestion on isolating pure RNA from mouse muscle tissues.
My 260/280 reading are 1.98 but 260/230 is very low.
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Hai,
You take 300 mg of tissue, cut and slice well. After that, you go for homogenate because, skeletal muscles are very hard. Then follow the QiagenRNEasy kit. You should wash properly in each step. Read at 260/280 nm. The ratio is 1.72 to 2.1.
You will get low ration means, run the Gel. Check the two bands.
After go for DNAse treatment.
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Hello,
I am wanting to express mouse skeletal muscle contractile performance to lean tissue mass, and I had been advised in a piece of peer review for a paper that it is possible to perform such an analyses by placing skeletal muscles into chloroform methanol for several days, drying, and expressing contractile performance to the remaining lean tissue mass. I am struggling to find anything in the literature to have used this approach for extracting lipids from mouse skeletal muscles.
Is there a published protocol regarding this analyses, or does anyone have a step-by-step guide they could direct me to? Or is it really as simple as the above procedure?
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Here is a quick methods paper describing lipid extraction from muscle tissue (human).
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Hi all,
I have been having issues with my samples not running cleanly during SDS PAGE. The samples come from fractionated skeletal muscle where the 'nuclear' samples are causing the issue whilst the 'cytosolic' samples are running cleanly through the gel. I have been using the Bio-Rad Mini TGX pre-cast gels and making fresh 1X TGS buffer running the samples slowly at 80-100V.
My initial thoughts is that it is the sample preparation as this is only occurring in the 'nuclear' samples where the 'cytosolic' samples are being run in the same tank.
Any advice would be much appreciated.
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Michael Plank Benzonase would be a solution, but depends on the next steps - it is a contaminating protein added to do the sample, so if this sample will be used e.g. for MS, then I would be very careful with adding any new protein.
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I search a protocol to extract Dna and rna at the same time from skeletal muscle. There are some kit like qiagen allprep but it seem that it doesn't work with skeletal muscle. Anyone have tried a kit for skeletal muscle or a specific protocol?
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Yi Wan
No it's the problem, my sample is really precious that's why i can use my classical protocol (Trizol for RNA, and special kit for DNA)
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We would like to test different AAV serotypes to transduce skeletal muscle cells from rat. In order to spare AAV we would like to make some test on adult cultured cells. FDB is easy to dissociate and allows the primary culture of fully differentiated skeletal muscle cells for several days. However litterature on about the use of AAV in these conditions is lacking.
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Hi Bodvael,
In my experience, I would suggest you to use AAV9 and AAV-DJ, which are good at in vitro cell infection. AAV-DJ is better in most of cell lines,but AAV9 may work better in primary cells.
Genemedi is expert in AAV production and genetherapy, you could find more information on this website: www.genemedi.net/i/aav-packaging
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I read a paper about treating muscular dystrophy patient with AAV9 virus infection as a carrier.
AAV9 is known to recognize muscle specifically.
Is there any mechanism that figured out how does AAV9 infect muscle??
Thanks.
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In general the tropism of AAV is determined by its capsid structure. The capsid of AAV9 preferentially binds to terminal N-linked galactose, a certain sugar structure which is present on the surface of a variety of cells (e.g. as part of glycoproteins). Additional protein(s) which serve as AAV9 receptor(s) need to be present on the cell membrane as well. One candidate is the transmembrane protein KIAA0319L (also termed AAVR). After binding to AAVR the AAVs are taken up by the cell via clathrin-mediated endocytosis togehter with AAVR. The amount of N-linked galactose in the cell membrane together with AAVR and/or other protein(s) that serve as AAV9 receptor(s) determines the amount of AAV9 being taken up by the cell. Skeletal muscle cells seem to present a substantial amount of these molecules on their surface. Keep in mind though, that AAV9 is not 100% specific for skeletal muscle cells and is also able to transduce a large number of different other cell types that also display the relevant AAV9 receptors.
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Hello,
I have a problem to find primary references showing what is happening with glucose after glucose load (postprandially, during glucose tolerance test, in hyperinsulinemic euglycemic clamp,..). It is usually assumed that insulin drives most of the glucose to skeletal muscle, but I cannot find any references really showing the comparison of individual tissues... Some references to PET studies or other radiotracer uptake studies would be really helpful.
Thank you all.
Ram
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You might find our article „redistribution of glucose from skeletal muscle to adipose tissue during catch-up fat“ published in Diabetes useful. The article can be downloaded from my profile and you can find there absolute values for glucose uptake in different tissues during a hyperinsulinemic-euglycemic clamp
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I am looking for studies comparing measures of skeletal muscle blood flow using CEU and gold standard imaging techniques in humans
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Hi Annelise, this is a very nice paper.
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