Has anyone a procedure of skeletal muscle fiber bundles mechanical separation recorded (respirometry, Oroboros)? I am looking for tips and tricks on that (separation techniques and final separated form). I cannot get a response after adding ADP during the protocol. It probably means that I damaged the fibers during the separation process or I do not separate them enough to get them permeabilized. Anyone?
Thanks.
I did not have any experience in this area, but I could recommended the methods use in the data of Professor Józef Langfort from our academy
best regards
Hi Robert,
How do you currently separate and permeabilize fibers? Also, what species are you studying?
Regardless, I would recommend watching this JoVE video for how to separate fibers: http://www.jove.com/video/2431/respirometric-oxidative-phosphorylation-assessment-saponin
The Oroboros website (http://oroboros.at) is also an excellent resource for both protocols and theories relating to respirometry.
Hi Robert
You should use a very good pair of forceps/tweezers to separate the fibres. If they're blunt, you are more likely to damage the fibres and therefore get very little response with the substrates you're using. Ideally aim to separate out into fibre bundles of approximately 3-5 fibres each - this allows the mitochondria to be maximally exposed without running the risk of individual fibres being damaged or separated. This is what I have found worked best for me with human skeletal muscle samples. Once you've separated out the fibres you should permeabilize them for 30 minutes in BIOPS solution (see Oroboros website) containing 50 μg/mL of Saponin. You should then wash the Saponin off by rinsing the sample twice for 5 minutes each in the respiration buffer you're using - we currently use Mir05. You could also try adding Blebbistatin to the chambers to prevent the samples from contracting.
You should look at the oroboros website for further advice - they have a lot of good information on there regarding protocols.
Hi Jack,
Good point. I think I press the fibers too much and sort of try to separate them by going with tweezers over it (like with your hand over grass). I guess what you mean is that I should go more in between the fibers with the sharp ends and separate down to one layer of groups of 3-5 fibers.
I also use 5-7 mg of tissue per chamber, and at this moment that makes it quite challenging to be accurate and quick. In accordance with the time, it takes me up to 30 min to work it out on a dish. How quick do you do this and what is the time impact on the respiration? I know there is, but is there a critical difference between 10 and 15 min?
Thanks so much for your feedback,
Best,
Robert.
Hi Robert
How are you getting on with the separation now? Yeah you should be able to get the tweezers in between the individual muscle fibres to separate them out in to bundles. I think 5-7mg is sufficient. You could try to go for less (I think Oroboros recommend 2-4mg). However, I prefer to use more tissue (~5-10mg). As the JO2 measurements in DatLab are calculated by dividing the decline in oxygen consumption over the amount of tissue, the less tissue you use means that the oxygen flux trace can be somewhat bouncy (especially if you are only using 2mg or less). Therefore I think you get a much better signal and smoother line with more tissue. However, this may require more reoxygenations of the chambers if you are performing the experiment under hyperoxic conditions.
I probably take a similar amount of time or perhaps a little less (~20mins) to dissect. I haven't actually tested to see if the time in the dish does make a difference but I suspect that, if it does have an effect, it's only minimal.
Best wishes,
Jack
Dear Robert:
To isolate muscle bundles from Quail pectoral muscle, the blood of muscle was washed out with saline solution through left ventricle of heart, followed by fixation with perfusion of Carnoy solution which was most effective to isolate single muscle fibers and/or muscle bundles than other fixations examined. Small muscle clumps were pinned on gelatin in Petri dish and isolated into as small bundle as possible with fine forceps under stereomicroscope whose lighting on sumples were from both under and upper. Many muscle fragments gathered in test tube, were washed by centrifuge with PBS two times. They were then incubated with PBS including collagenase for 30 min at 37 C. followed by washing with PBS two times to remove enzyme. Fragments enzymatically digested, were stained with hematoxylin staining solution and washed three times with PBS to remove excess pigments. Fragments on the bottom of test tube were mixed with diluted gelatin and centrifuged x30,00 once. Small fragments were taken together from upper part of test tube with fine pipet, while larger ones placed on the bottom of tube. They were mounted on slide grasses to observe under microscope.
Hope suggestive for you.
Tateki Kikuchi
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