THRIFTY: a novel high‐throughput method for rapid fibre type identification of isolated skeletal muscle fibres

Abstract Fibre type‐specific analyses are required for broader understanding of muscle physiology, but such analyses are difficult to conduct due to the extreme time requirements of dissecting and fibre typing individual fibres. Investigations are often confined to a small number of fibres from few participants with low representativeness of the entire fibre population and the participant population. To increase the feasibility of conducting large‐scale fibre type‐specific studies, a valid and rapid method for high‐throughput fibre typing of individually dissected fibres was developed and named THRIFTY (for high‐THRoughput Immunofluorescence Fibre TYping). Employing THRIFTY, 400 fibre segments were fixed onto microscope slides with a pre‐printed coordinated grid system, probed with antibodies against myosin heavy chain (MyHC)‐I and MyHC‐II and classified using a fluorescence microscope. The validity and speed of THRIFTY was compared to a previously validated protocol (dot blot) on a fibre‐to‐fibre basis. Fibre pool purity was evaluated using ‘gold standard’ SDS‐PAGE and silver staining. A modified THRIFTY‐protocol using fluorescence western blot equipment was also validated. THRIFTY displayed excellent agreement with the dot blot protocol, κ = 0.955 (95% CI: 0.928, 0.982), P < 0.001. Both the original and modified THRIFTY protocols generated type I and type II fibre pools of absolute purity. Using THRIFTY, 400 fibres were typed just under 11 h, which was approximately 3 times faster than dot blot. THRIFTY is a novel and valid method with high versatility for very rapid fibre typing of individual fibres. THRIFTY can therefore facilitate the generation of large fibre pools for more extensive mechanistic studies into skeletal muscle physiology. Key points Skeletal muscle is composed of different fibre types, each with distinct physiological properties. To fully understand how skeletal muscle adapts to external cues such as exercise, nutrition and ageing, fibre type‐specific investigations are required. Such investigations are very difficult to conduct due to the extreme time requirements related to classifying individually isolated muscle fibres. To bypass this issue, we have developed a rapid and reliable method named THRIFTY which is cheap as well as versatile and which can easily be implemented in most laboratories. THRIFTY increases the feasibility of conducting larger fibre type‐specific studies and enables time‐sensitive assays where measurements need to be carried out in close connection with tissue sampling. By using THRIFTY, new insights into fibre type‐specific muscle physiology can be gained which may have broad implications in health and disease.

1. The authors highlight that SDS-PAGE is the gold standard and is used in their work to help validate the THRIFTY approach. The comment on page 17 about contamination from the dissection process is a minor concern and seems exaggerated as a reason not to use the gold standard approach. There are various solutions that are viable for isolating muscle fibers (RNAlater, RNA-best, etc.) that make isolation quite easy, reliable, and provide opportunities for various applications (fiber type, fiber pooling, protein identification, molecular probing). In contrast the freeze-dried muscle fiber isolation can be rather labor some and also requires a special skillset. Thus, it would depend upon the research question and approach to which technique might be most applicable.
2. The Murach 2016 paper studied resistance trained individuals (presented on page 16), which given their training status would be expected to have few hybrid (<10%) muscle fibers. However, the amount of hybrid fibers can easily scale to >20% or more with inactivity and prolonged disuse, which may alter the fidelity of small fiber populations to accurately reflect the fiber type profile.

Supplemental Figures 2 & 3 are not totally visible and appear to be cropped off the page.
4. Cost is mentioned for the slides and printing grid but is a mention of the cost of the CELENA S Digital Imaging System worth it as these microscopes can be a huge expense to consider to be able to do THRIFTY. Something you don't need for SDS-PAGE.
5. The statement on how hybrids are visually determined in THRIFTY (page 18, line 2) would be better placed in the methods section.
7. Page 5: lowercase "western blot" to keep consistent. Apply to rest of paper.
8. Page 11: To give context to inconclusive THRIFTY hybrid and dual fibre segments (Seven fibre segments were deemed inconclusive.....) consider referring the reader to Figure 3 for a visual example. 9. Page 12: Change "se" to "see" on 3rd line of last paragraph.
10. Page 16: missing parenthesis at the end first discussion paragraph.
11. Page 28: Supplemental Figure 3 legend is incorrectly labeled " Figure S2." Referee #2: The authors describe a proposed new method (with the acronym THRIFTY) for a more rapid identification of MyHC isoform content (type 1 vs type II)in single fiber segments from human biopsy samples. The technique involves the dissection of single fiber segments from a biopsy samples, the arrangement of these individual fiber segments onto a grid on a microscope slide and then using currently existing and well used techniques (primary AB to MyHC-I and MyHC-II, followed by fluorescently labelled second Ab and use of a digital fluorescent microscope) to visualize these fibers and their MyHC isoform content.
While the technique is well described and is somewhat validated against some other existing techniques, I have the following concerns:

08-Feb-2022
1 -It appears that the only really novel contribution of this technique is the grid pattern printed onto the microscope slide. All other aspects of this technique are well developed and highly used currently in the field.
2-The authors correctly point out that identification of fiber type (or MyHC isoform phenotype) is a painstaking process that often limits the number of fibers that can be tested. However, it is not obvious to this reviewer how this new technique significantly alters the painstaking nature of this process. The limiting factor of this technique remains the dissection of hundreds of individual fibers from biopsy samples. Even though this proposed technique may involve simpler or faster steps after the dissection -compared to dot blot or SDS-PAGE -the need to individually dissect fibers from the sample keeps this technique from being a true "high-throughput technique". Several published papers have done MyHC isoform analysis of more than 1000 fibers using SDS-PAGE analysis.
3-The paper would strongly benefit from some examples of how this technique might be used to better answer questions of physiological significance compared to other existing techniques of single fiber identification. Even better would be for this technique to be used within this paper to answer a physiological question, in order to show the power of this technique. For example, if the question to be answered is simply how does a given intervention or condition affect fiber type (as given by MyHC isoform) distribution in a muscle, this is much more quickly and completely answered using immunohistochemistry on cross-sections of muscle. With IHC on cross-sections one can visualize MyHC isoform content of all fibers in a cross-section immediately with no need for painstaking fiber dissection. If the point is for the THIFTY method to be able to connect the MyHC isoform content of a fiber to some other measurement in that fiber (contractile function, etc.) then it is likely that this other measurement would be the limiting factor in throughput and therefore the small amount of time gained with the THRIFTY method would be inconsequential in improving throughput. Without seeing the THRIFTY method actually used to answer a physiological question it is difficult to gauge its importance.
4-The authors compare the THRIFTY results to SDS-PAGE analysis of MyHC isoform content as a way of validating the THRIFTY technique, but make the curious choice to pool all samples identified as a particular type and run those on a SDS-PAGE gel. The true power of the SDS-PAGE analysis of single fiber MyHC isoform content is the ability to determine MyHC isoforms in individual fibers. The proper validity comparison is to compare the SDS-PAGE results from a large number of individual fiber segments with the results given by the THRIFTY analysis of those same fibers, thus showing that the THRIFTY method has the same level of accuracy as SDS-PAGE for on an individual fiber identification. ****************************** Confidential Review 25-Apr-2022 1st Authors' Response to Referees

22-Jun-2022 1st Revision -Editorial Decision
Dear Dr Apró, Re: JP-TFP-2022-283262X "THRIFTY, a novel high-throughput method for rapid fibre type identification of isolated skeletal muscle fibres" by Oscar Horwath, Sebastian Edman, Alva Andersson, and William Apró Your appeal to the recent decision on your Journal of Physiology manuscript has been carefully considered by both the review editor and the senior editor. I am pleased to inform you that your appeal has been accepted and therefore, I would like to provide you with one final opportunity to revise your report for further review. Please note that this acceptance of your appeal does NOT guarantee acceptance of the revised paper. Indeed, a final decision about acceptance will be made after re-review and further evaluation by the review editor. Thank you for your patience with this process. We look forward to receiving your revised report.
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----------------EDITOR COMMENTS Reviewing Editor: While the 2 new reviewers of this manuscript felt that your study has significant merit, both reviewers felt that more experiments would be necessary to push this study into being of great interest to the readership of the Journal of Physiology. We are sorry that this has been an extended review process, but the 2 new reviewers did not provide any additional support for further consideration of your study.

Senior Editor:
Your appeal to the recent decision on your Journal of Physiology manuscript has been carefully considered by both the review editor and the senior editor. I am pleased to inform you that your appeal has been accepted and therefore, I would like to provide you with one final opportunity to revise your report for further review. Please note that this acceptance of your appeal does NOT guarantee acceptance of the revised paper. Indeed, a final decision about acceptance will be made after re-review and further evaluation by the review editor. Thank you for your patience with this process. We look forward to receiving your revised report.
No statistics summary has been submitted.
This is a revised (R1) paper that is improved over the first submission. The additions to the manuscript add clarity and also provide a better scientific foundation for the THRIFTY approach. The newly included "Applications" sections highlights in better detail the ability of the THRIFTY protocol to run specific assays at the fibre type level (i.e. metabolites, mass spec, PCR, proteomics, etc.). Additionally, Figure 4 and Supplement Figure 4 provide greater detail into the THRIFTY protocols fiber type specific applications and some data from the method.
The muscle glycogen content shown in slow and fast muscle fibers is on the high end (1000 mmol/kg dry wt) compared to previous literature in this area (Essen et al., Acta Physiol Scand 95:153-165, 1975 andGreenhaff et al., J Physiol, 478: 149-155, 1984). Please comment.
Referee #3: In this manuscript, the authors describe an improved protocol for rapid fiber the determination on isolated muscle fiber segments. Overall, the work was conducted carefully and the article is well-written. I appreciate the effort put into this work and think the method has the potential to be adopted by the skeletal muscle field. I think the authors could do a better job of describing how most aspects of fiber typing single fibers is a "fixed cost" from a time perspective. For instance, isolating individual fibers is tedious no matter who is doing it; for even for the most skilled technician, this is going to take time (i.e. is more-or-less fixed cost). Where THRIFTY obviously shines is the actual fibertyping. Where single fiber SDS-PAGE may take 18-24 hours, the THRIFTY technique only takes a few hours. This time savings opens up all kinds of opportunities that weren't fully explored in this manuscript (which the authors highlight in the last paragraph). I think this point could also be reinforced in the Key Points at the beginning of the manuscript.
In order to fully realize the potential of the THRIFTY technique, the authors should perform respirometry on "wet" slow and fast muscle fibers from humans. The THRIFTY technique will make this possible since those types of experiments must be performed "fresh", or within a matter of hours from tissue collection. This could be an n=1 proof of concept experiment, but would drive home how impactful this technique could be for the field, as well as provide some novel information on fiber type-specific metabolism in humans.
Minor comments: 1. Contamination for sfSDS-PAGE only really occurs when isolating fibers from non-skinned bundles. Granted, that is what is being done here, but I think it is worth differentiating the two.
2. What was the rationale for the antibody dilutions? Also, why use supernatant for the myosin antibodies when concentrate gives you greater control over the amount of antibody being applied?
3. Is a "mailer" analogous to a Coplin jar? Perhaps make that clear.
In Figure 4, I would put a total time estimate somewhere on the Figure. It also seems like this figure could serve as the graphical abstract, perhaps with some additional information on what make the technique advantageous over others?
Referee #4: The THRIFTY method presented adds some advantage to current published methodologies. The most impressive is that fibres collected are not required to be denatured and so they may be used for measurements other than western blotting. This is a welcomed addition. There are many factors, however, that do not work in the favor of THRIFTY. For instance, the time required for collecting the high number of fibres is no different and is the most consuming step present. As acknowledged in the manuscript, technician experience will dictate the length of this (and other) steps. Other steps may be quicker, but what about the trade-off which is that the single fibres are left at room temperature for a long period whilst fibre typing is taking place. This is particularly problematic if they are not kept in a (denaturing) solution because there may be proteolytic activity occurring during this time. Freeze dried fiber segments are small and may absorb water from the air, which could initiate these events. Whilst the lack of requiring labelling tubes saves time, it may introduce a confounding factor. This would need to be addressed.
Assumably if 1000 fibres are collected to be analysed together, for the cost-time benefit outlined, what validation has been done to ensure that the fibre collection time does not affect downstream steps and measurements?
One of the issues in single fiber methodology is the lack of being able to simply and accurately detect MHC IIx fibers in humans. In this manuscript, this issue remains, however as pointed out by the authors, in muscle samples from healthy individuals this number would be low and so remain out of scope. For a similar reason, the number and type of hybrid fibers that are present, whilst with THRIFTY can be observed (assuming the microscopy method is used), yet once again the number would be low and so downstream measurements would be unlikely. As such, there is no benefit of THRIFTY to add these missing steps for single fiber analyses.
Page 17: "Given these large variations, pooling too few fibres may prevent proper identification of mechanistic links between different variables." And "..... the pool should consist of as many fibres as possible." Variation is an issue, however a number of fibers should be provided, not 'as many as possible'. One could go on collecting fibers for a very long time, so the number that 'is possible' is very high. Choosing '100' is random, some analyses should be done to arrive at the number of fibres required.
Page 23: "high resolution mitochondrial respirometry" would require fresh tissue, how could the fibres be collected, fiber typed and then used for measurements? This would still be a delay of some hours. In order to make this statement, data should be provided.

25-Apr-2022
Fig S4: appears to show data for a single set of type I or type II fibers. This is not representative, and would need a higher 'n' for each measure and statistics to be included.

_______________________________________________ END OF COMMENTS
1st Confidential Review

----------------EDITOR COMMENTS
Reviewing Editor: While the 2 new reviewers of this manuscript felt that your study has significant merit, both reviewers felt that more experiments would be necessary to push this study into being of great interest to the readership of the Journal of Physiology. We are sorry that this has been an extended review process, but the 2 new reviewers did not provide any additional support for further consideration of your study.

Senior Editor:
Your appeal to the recent decision on your Journal of Physiology manuscript has been carefully considered by both the review editor and the senior editor. I am pleased to inform you that your appeal has been accepted and therefore, I would like to provide you with one final opportunity to revise your report for further review. Please note that this acceptance of your appeal does NOT guarantee acceptance of the revised paper. Indeed, a final decision about acceptance will be made after re-review and further evaluation by the review editor. Thank you for your patience with this process. We look forward to receiving your revised report.
No statistics summary has been submitted.
Thank you for the opportunity to submit av revised version of our manuscript. We have performed the requested analyses and we have included a statistics summary. -----------------

REFEREE COMMENTS
Referee #1: This is a revised (R1) paper that is improved over the first submission. The additions to the manuscript add clarity and also provide a better scientific foundation for the THRIFTY approach. The newly included "Applications" sections highlights in better detail the ability of the THRIFTY protocol to run specific assays at the fibre type level (i.e. metabolites, mass spec, PCR, proteomics, etc.). Additionally, Figure 4 and Supplement Figure 4 provide greater detail into the THRIFTY protocols fiber type specific applications and some data from the method.
Thank you for taking the time to once again review or manuscript and for the constructive comments.
The muscle glycogen content shown in slow and fast muscle fibers is on the high end (1000 mmol/kg dry wt) compared to previous literature in this area (Essen et al., Acta Physiol Scand 95:153-165, 1975 andGreenhaff et al., J Physiol, 478: 149-155, 1984). Please comment.
In the ongoing study from which the fibres were dissected we used a protocol consisting of a bilateral glycogen depletion session to maximize subsequent glycogen repletion. This was followed by 2.5 days of carbohydrate loading with 10 g of carbohydrate per kg body weight. Approximately 35% of the carbohydrates were supplied as a glucose polymer with very high molecular mass which has been shown to enhance glycogen resynthesis (Piehl-Aulin et al. Eur J Appl Physiol. 2000 Mar;81(4):346-51). As such the high values are expected and similar values can be found in the literature. For instance, in the paper by Essen and Henriksson (Acta Physiol Scand. 1974 Mar;90(3):645-7.) the highest reported value is 978 mmol/kg after glycogen depletion and subsequent replenishment, and resting values of 881 mmol/kg are reported by Essén et el. (Acta Physiol Scand 95:153-165, 1975). Similar values (> 900 mmol/kg) have also been reported on whole muscle (Wojtaszewski et al. Am J Physiol Endocrinol Metab. 2003 Apr;284(4):E813-22.).

Referee #3:
In this manuscript, the authors describe an improved protocol for rapid fiber the determination on isolated muscle fiber segments. Overall, the work was conducted carefully and the article is wellwritten. I appreciate the effort put into this work and think the method has the potential to be adopted by the skeletal muscle field.
Thank you for taking the time to review our manuscript and for providing constructive comments.
I think the authors could do a better job of describing how most aspects of fiber typing single fibers is a "fixed cost" from a time perspective. For instance, isolating individual fibers is tedious no matter who is doing it; for even for the most skilled technician, this is going to take time (i.e. is more-or-less fixed cost). Where THRIFTY obviously shines is the actual fiber-typing. Where single fiber SDS-PAGE may take 18-24 hours, the THRIFTY technique only takes a few hours. This time savings opens up all kinds of opportunities that weren't fully explored in this manuscript (which the authors highlight in the last paragraph). I think this point could also be reinforced in the Key Points at the beginning of the manuscript.
We have now added a more extended description of the fixed time cost in the introduction as well as an expanded discussion about the potential implications of the time savings when using THRIFTY. We have also emphasized this in an additional Key Point.
In order to fully realize the potential of the THRIFTY technique, the authors should perform respirometry on "wet" slow and fast muscle fibers from humans. The THRIFTY technique will make this possible since those types of experiments must be performed "fresh", or within a matter of hours from tissue collection. This could be an n=1 proof of concept experiment, but would drive home how impactful this technique could be for the field, as well as provide some novel information on fiber type-specific metabolism in humans.
We have now performed respirometry on freshly dissected type I and type II from one individual and have included it in the manuscript. Please see figure 10.
Minor comments: 1. Contamination for sfSDS-PAGE only really occurs when isolating fibers from non-skinned bundles. Granted, that is what is being done here, but I think it is worth differentiating the two. Point taken; this distinction has now been added to the manuscript.
2. What was the rationale for the antibody dilutions? Also, why use supernatant for the myosin antibodies when concentrate gives you greater control over the amount of antibody being applied?
Before the development of THRIFTY, we had been using supernatant for fibre typing muscle crosssections and therefore had the supernatant antibodies readily available for pilot experiments. Based purely on laboratory practice, pilot experiments were initiated with BA-F8 and SC-71 at 1:1000 but the signal was weaker for SC-71 so the concentration was increased to 1:500 which produced a satisfactory signal.
Based on the information from DSHB, one vial of concentrate contains the same amount of IgG as one vial of supernatant (if produced from the same batch) but in ten times less volume which has been obtained by using concentration filters. It is therefore easy to adjust the volume of the antibody required from ones preferred antibody format. For subsequent typing we have used concentrate instead but at ten-fold lower volumes with no difference in performance.
3. Is a "mailer" analogous to a Coplin jar? Perhaps make that clear.
Yes it is; this has been clarified in the manuscript.
In Figure 4, I would put a total time estimate somewhere on the Figure. It also seems like this figure could serve as the graphical abstract, perhaps with some additional information on what make the technique advantageous over others?
The figure has been changed accordingly. We have also included a graphical abstract with the reviewer's suggestions.

Referee #4:
The THRIFTY method presented adds some advantage to current published methodologies. The most impressive is that fibres collected are not required to be denatured and so they may be used for measurements other than western blotting. This is a welcomed addition.
Thank you for taking the time to review our manuscript and for acknowledging some of the benefits of THRIFTY.
There are many factors, however, that do not work in the favor of THRIFTY. For instance, the time required for collecting the high number of fibres is no different and is the most consuming step present. As acknowledged in the manuscript, technician experience will dictate the length of this (and other) steps.
We fully acknowledge that THRIFTY does not increase the speed of dissection. However, we make no such claim, and the issue of time-consuming dissection remains for all available methods used for single fibre typing. To state that this is a factor specifically not in favor of THRIFTY therefore seems unjustified and unfair.
Skill and experience will always be a factor to consider with all methods. That is why a technician inexperienced in both THRIFTY and dot blot performed all steps for both methods. In our comparison, the lack of experience effectively removes skill as a determining factor, and we show that THRIFTY is three times faster. It is therefore difficult to understand how this can be seen as anything other than a factor in favor of THRIFTY.
Other steps may be quicker, but what about the trade-off which is that the single fibres are left at room temperature for a long period whilst fibre typing is taking place. This is particularly problematic if they are not kept in a (denaturing) solution because there may be proteolytic activity occurring during this time. Freeze dried fiber segments are small and may absorb water from the air, which could initiate these events. Whilst the lack of requiring labelling tubes saves time, it may introduce a confounding factor. This would need to be addressed.
Please note that each individual step in THRIFTY is different from the steps in the dot blot after cutting of the minimal fibre segment to be used for typing, and the number of steps differ between the two methods. One should therefore refrain from trying to compare individual steps and instead look at the performance of the whole method from start to finish. As mentioned before, from start to finish, THRIFTY is considerably quicker.
The reviewer makes a good point regarding the potential trade-off of fibres being in room temperature and the risk of inducing proteolysis due to moisture in the air. It is reasonable to expect that freeze-dried tissue may absorb water from the air due to condensation when moved from the freezer to room temperature. However, the amount of water absorbed is minimal and should have minimal to no effect on biological processes such as proteolysis.
According to Lowry and Passonneau (1972, A flexible system of enzymatic analysis, Academic Press, New York), 1% of water is absorbed for each 10% humidity at ambient room temperature. Standard procedure in our laboratory when working with freeze-dried muscle is to maintain humidity below 40% and therefore a maximal increase in fibre weight of less than 4% could be expected due to absorption of water from the air. Equilibration occurs within minutes (Lowry and Passonneau, 1972) after which there should be no further increase in fibre weight. This has been confirmed by Essén et al in their early studies (Acta Physiol Scand. 1974 Mar;90(3):645-7 and Acta Physiol Scand. 1975, 95:153-165,) in which they weighed fibres repeatedly during the day and saw no increase in fibre weight.
In our laboratory, fibre dissection is always carried out below 40% humidity. We also take additional precautionary measures by always thawing freeze-dried muscle on moisture-absorbing silica-gel for at least 30 minutes prior to dissection. Thawing on silica-gel minimizes condensation which further reduces absorption of water. In our hands, when treated as above, fibre weight increases less than 0.3 % when left out for 24 hours at 21°C and 32% humidity. Given this extremely small increase, there is no reason to expect an increase in proteolysis.
To illustrate this, we compared three experimental conditions. 1: Immediate homogenization after thawing on silica-gel for 30 minutes. Bare minimum exposure to ambient room temperature and low humidity and therefore served as the control condition.
2: Homogenization after 24h at 21°C and 32% humidity. This is double the time of the THRIFTY protocol presented in the manuscript.
3: Homogenization after rehydration in water for 25 min at 21°C. Represents an extreme situation where freeze-dried muscle re-absorbs all the water previously removed during freeze-drying.
As can be seen in the example blots, there is no difference between the control sample and the sample left out for 24h. In contrast, proteolysis is clearly evident in the rehydrated samples.
We are therefore fully confident that there is no trade-off when using THRIFTY.
Assumably if 1000 fibres are collected to be analysed together, for the cost-time benefit outlined, what validation has been done to ensure that the fibre collection time does not affect downstream steps and measurements?
We are not fully clear on what the reviewer means here, but we never collect 1000 fibres at a time, nor do we state that in the manuscript. Fibre collection occurs prior to typing and the number of fibres collected during a day depends on several aspects, like the quality of the muscle biopsy and the skill of the technician. However, this applies to all methods and therefore has no relevance for assessing THRIFTY as a fibre typing method. Once fibres are ready for typing, THRIFTY is faster than dot blot independent of how many fibres were collected at the same time.
For clarification, what we state in the manuscript is that a skilled technician can type 800-1000 fibres under less than 11 hours. There is no rationale to expect any negative effects on downstream applications within this time, or even twice the time (see above), at least when using freeze-dried muscle fibres, as virtually no water is absorbed when fibres are handled according to standard procedure. This is evident from the proof-of-concept data included in the manuscript which all show expected differences and values within physiological range (i.e. higher cs activity and mitochondrial protein content in type I fibres, and lower glycogen levels in fibres from depleted muscle compared to fibres from a loaded muscle).
Also, if one has concerns about time-dependent negative effects for certain downstream measurements when using THRIFTY, it is easy to reduce the time by simply typing fewer fibres. There is no requirement to type 1000 fibres at the same time and THRFITY still outperforms other contemporary methods when it comes to speed.
One of the issues in single fiber methodology is the lack of being able to simply and accurately detect MHC IIx fibers in humans. In this manuscript, this issue remains, however as pointed out by the authors, in muscle samples from healthy individuals this number would be low and so remain out of scope. For a similar reason, the number and type of hybrid fibers that are present, whilst with THRIFTY can be observed (assuming the microscopy method is used), yet once again the number would be low and so downstream measurements would be unlikely. As such, there is no benefit of THRIFTY to add these missing steps for single fiber analyses.
We fully acknowledge that THRIFTY has not been validated for type IIX fibres. However, we respectfully disagree with the reviewer's statement about THRIFTY not adding any value for measurements on hybrid fibres.
THRIFTY is currently the only method that can identify hybrid fibres with certainty as it does not rely on denatured samples. Under the denatured conditions used for SDS-PAGE, single fibre western blot and dot blot, it is impossible to distinguish true hybrid fibres from fibres contaminated by the opposing fibre type. This issue is eliminated with THRIFTY. As such, THRIFTY does in fact add great value with its improved accuracy for researchers interested in measurements on hybrid fibres.
Page 17: "Given these large variations, pooling too few fibres may prevent proper identification of mechanistic links between different variables." And "..... the pool should consist of as many fibres as possible." Variation is an issue, however a number of fibers should be provided, not 'as many as possible'. One could go on collecting fibers for a very long time, so the number that 'is possible' is very high. Choosing '100' is random, some analyses should be done to arrive at the number of fibres required.
We agree with the reviewer's point that the number that is possible can be very high. However, this is a very general statement where we are trying to make the point that that in general terms, a large number of fibres will always be more representative than very few fibres, especially when there is uncertainty about the number of fibres required.
Providing such numbers in the manuscript is far beyond the current scope but we have rephrased this paragraph for more nuance. We do also provide several references for different variables in which variation has been noted, which can be used as starting points for researchers wanting to establish the minimum number of fibres required for their specific analyses.
We used 100 fibres of each type to prepare the control slide for the modified THRIFTY protocol to be used with the western blot equipment, but we do not state anywhere that 100 fibres should be used for subsequent analyses.
Page 23: "high resolution mitochondrial respirometry" would require fresh tissue, how could the fibres be collected, fiber typed and then used for measurements? This would still be a delay of some hours. In order to make this statement, data should be provided.
We have now performed respirometry measurements on pools of type I and type II fibres from freshly collected muscle. Please see figure 10.
Dissection, typing and pooling took approximately 6 hours and within this time frame there is no significant time effect on respiration when muscle is kept in cold preservation media (Cardinale et al. Physiol Rep. 2018 Feb;6(4):e13611) Fig S4: appears to show data for a single set of type I or type II fibers. This is not representative, and would need a higher 'n' for each measure and statistics to be included.
The data presented are not meant to show any effects of an intervention but are only for proof-ofconcept to show the strength of THRIFTY in generating large pools of fibres for a wide range of subsequent analyses.