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Biology

Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry

Published: December 01, 2023
doi:
10.3791/65944
, , ,
1Department of Biomedicine,Aarhus University, 2Aarhus Institute of Advanced Studies,Aarhus University

Summary

The protocol presented here enables the identification and high-dimensional analysis of muscle stem and progenitor cells by single-cell mass cytometry and their purification by FACS for in-depth studies of their function. This approach can be applied to study regeneration dynamics in disease models and test the efficacy of pharmacological interventions.

Abstract

Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle stem cells become activated upon muscle injury. Following activation, they proliferate, and most of their progeny differentiate to generate fusion-competent muscle cells while the remaining self-renews to replenish the stem cell pool. While the identity of muscle stem cells was defined more than a decade ago, based on the co-expression of cell surface markers, myogenic progenitors were identified only recently using high-dimensional single-cell approaches. Here, we present a single-cell mass cytometry (cytometry by time of flight [CyTOF]) method to analyze stem cells and progenitor cells in acute muscle injury to resolve the cellular and molecular dynamics that unfold during muscle regeneration. This approach is based on the simultaneous detection of novel cell surface markers and key myogenic transcription factors whose dynamic expression enables the identification of activated stem cells and progenitor cell populations that represent landmarks of myogenesis. Importantly, a sorting strategy based on detecting cell surface markers CD9 and CD104 is described, enabling prospective isolation of muscle stem and progenitor cells using fluorescence-activated cell sorting (FACS) for in-depth studies of their function. Muscle progenitor cells provide a critical missing link to study the control of muscle stem cell fate, identify novel therapeutic targets for muscle diseases, and develop cell therapy applications for regenerative medicine. The approach presented here can be applied to study muscle stem and progenitor cells in vivo in response to perturbations, such as pharmacological interventions targeting specific signaling pathways. It can also be used to investigate the dynamics of muscle stem and progenitor cells in animal models of muscle diseases, advancing our understanding of stem cell diseases and accelerating the development of therapies.

Introduction

Skeletal muscle constitutes the largest tissue by mass in the body and regulates multiple functions, from eyesight to respiration, from posture to movement, as well as metabolism1. Therefore, maintaining skeletal muscle integrity and function is critical to health. Skeletal muscle tissue, which consists of tightly packed bundles of multinucleated myofibers surrounded by a complex network of nerves and blood vessels, exhibits remarkable regenerative potential1,2.

The main drivers of skeletal muscle regeneration are adult muscle stem cells (MuSCs). Also known as satellite cells, due to their unique anatomical location adjacent to the plasma membrane of the myofiber and beneath the basal lamina, they were first identified in 19613. MuSCs express a unique molecular marker, the transcription factor paired box 7 (Pax7)4. Mostly quiescent in healthy adults, they become activated upon muscle injury and proliferate to give rise to progeny that will (i) differentiate into fusion-competent muscle cells that will form new myofibers to repair muscle damage or (ii) self-renew to replenish the stem cell pool5.

At the cellular and molecular level, the process of regeneration is quite dynamic and involves cell-state transitions, characterized by the coordinated expression of key myogenic transcription factors, also known as myogenic regulatory factors (MRFs)6,7. Prior in vivo developmental studies, lineage tracing experiments, and cell culture work using myoblasts have shown that sequential expression of these transcription factors drives myogenesis, with myogenic factor 5 (Myf5) being expressed upon activation, myogenic differentiation 1 (MyoD1) expression marking commitment to the myogenic program, and myogenin (MyoG) expression marking differentiation8,9,10,11,12,13,14. Despite this knowledge and the discovery of cell surface markers to purify MuSCs, strategies and tools to identify and isolate discrete populations along the myogenic differentiation path and resolve a myogenic progression in vivo have been lacking15,16,17,18.

Here, we present a novel method, based on recently published research, which enables the identification of stem and progenitor cells in skeletal muscle and the analysis of their cellular, molecular, and proliferation dynamics in the context of acute muscle injury19. This approach relies on single-cell mass cytometry (also known as Cytometry by Time of Flight [CyTOF]) to simultaneously detect key cell surface markers (α7 integrin, CD9, CD44, CD98, and CD104), intracellular myogenic transcription factors (Pax7, Myf5, MyoD, and MyoG) and a nucleoside analog (5-Iodo-2′-deoxyuridine, IdU), to monitor cells in S phase19,20,21,22,23. Moreover, the protocol presents a strategy based on the detection of two cell surface markers, CD9 and CD104, to purify these cell populations by fluorescence-activated cell sorting (FACS), therefore enabling future in-depth studies of their function in the context of injury and muscle diseases. While primary myoblasts have been extensively used in the past to study the late stages of myogenic differentiation in vitro, it is not known whether they recapitulate the molecular state of muscle progenitor cells found in vivo24,25,26,27,28,29,30. The production of myoblasts is laborious and time-consuming, and the molecular state of this primary culture changes rapidly upon passaging31. Hence, freshly isolated myogenic progenitors purified with this method will provide a more physiological system to study myogenesis and the effect of genetic or pharmacological manipulations ex-vivo.

The protocol presented here can be applied to address a variety of research questions, for example, to study the dynamics of the myogenic compartment in vivo in animal models of muscle diseases, in response to acute genetic manipulations or upon pharmacological interventions, therefore deepening our understanding of muscle stem cell dysfunction in different biological contexts and facilitating the development of novel therapeutic interventions.

Protocol

Animal procedures were approved by the Danish animal experiments inspectorate (protocol # 2022-15-0201-01293), and experiments were performed in compliance with the institutional guidelines of Aarhus University. Analgesia (buprenorphine) is provided in drinking water 24 h prior to injury for the mice to adapt to the taste. Supplying buprenorphine in drinking water is continued for 24 h post-injury. Together with a subcutaneous (s.c.) injection of buprenorphine at the time of acute muscle injury, buprenorphine in the drin…

Representative Results

Here we present an overview of the experimental setup for using this combined approach which includes (i) high-dimensional CyTOF analysis of an acute injury time course by notexin injection to study the cellular and molecular dynamics of stem and progenitor cells in skeletal muscle (Figure 1, top scheme); and (ii) FACS of stem and progenitor cells using two cell surface markers, CD9 and CD104, to isolate these populations and perform in-depth studies of their function (F…

Discussion

Skeletal muscle regeneration is a dynamic process that relies on the function of adult stem cells. While prior studies have focused on the role of muscle stem cells during regeneration, their progeny in vivo has been understudied, primarily due to a lack of tools to identify and isolate these cell populations15,16,17,18. Here, we present a method to simultaneously identify and isolate …

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank the members of the FACS Core Facility in the Department of Biomedicine at Aarhus University for technical support. We thank Alexander Schmitz, the manager of the Mass Cytometry Unit at the Department of Biomedicine, for discussion and support. Scientific Illustrations were created using Biorender.com. This work was funded by an Aarhus Universitets Forskningsfond (AUFF) Starting Grant and a Start Package grant (0071113) from Novo Nordisk Foundation to E.P.

Materials

15 mL centrifuge tube Fisher Scientific 07-200-886
20 G needle KDM KD-fine 900123
28 G, 0.5 mL insulin syringe  BD 329461
29 G, 0.3 mL insulin syringe BD 324702
3 mL syringes Terumo medical MDSS03SE
40 µm cell strainers Fisher Scientific 11587522
5 mL polypropylene tubes  Fisher Scientific 352002
5 mL polystyrene test tubes with 35 µm cell strainer Falcon 352235
5 mL syringes Terumo medical SS05LE1
50 mL centrifuge tube Fisher Scientific 05-539-13
5-Iodo-2-deoxyuridine (IdU) Merck I7125-5g
anti-CD104 FITC (clone: 346-11A) Biolegend 123605 Stock = 0.5 mg/mL
anti-CD11b APC-Cy7 (Clone: M1/70) Biolegend 101226 Stock = 0.2 mg/mL
anti-CD31 APC-Cy7 (clone: 390) Biolegend 102440 Stock = 0.2 mg/mL
anti-CD45 APC-Cy7 (Clone: 30-F11) Biolegend 103116 Stock = 0.2 mg/mL
anti-CD9 APC (clone: KMC8) ThermoFisher Scientific 17-0091-82 Stock = 0.2 mg/mL
anti-Sca1 (Ly6A/E) APC-Cy7 (clone: D7) Biolegend 108126 Stock = 0.2 mg/mL
anti-α7 integrin PE (clone: R2F2)) UBC AbLab 67-0010-05 Stock = 1 mg/mL
BD FACS Aria III (4 laser) instrument BD Biosciences N/A 405, 488, 561, and 633 nm laser
Bovine Serum Albumin Sigma Aldrich A7030-50G
Buprenorphine 0.3 mg/mL Ceva Vnr 054594
CD104 (Clone: 346-11A) BD Biosciences 553745 Dy162; In-house conjugated
CD106/VCAM-1 (Clone: 429 MVCAM.A) Biolegend 105701 Er170; In-house conjugated
CD11b (Clone: M1/70) BD Biosciences 553308 Nd148; In-house conjugated
CD29/Integrin β1 (Clone: 9EG7) BD Biosciences 553715 Tm169; In-house conjugated
CD31 (Clone: MEC 13.3) BD Biosciences 557355 Sm154; In-house conjugated
CD34 (Clone: RAM34) BD Biosciences 551387 Lu175; In-house conjugated
CD44 (Clone: IM7) BD Biosciences 550538 Yb171; In-house conjugated
CD45 (Clone: MEC 30-F11) BD Biosciences 550539 Sm147; In-house conjugated
CD9 (Clone: KMC8) Thermo Fisher Scientific 14-0091-85 Yb174; In-house conjugated
CD90.2/Thy1.2 (Clone: 30-H12) BD Biosciences 553009 Nd144; In-house conjugated
CD98 (Clone: H202-141) BD Biosciences 557479 Pr141; In-house conjugated
Cell Acquisition Solution/Maxpar CAS-buffer Standard Biotools 201240
Cell-ID Intercalator-Iridium Standard Biotools 201192B cationic nucleic acid intercalator
Cisplatin Merck P4394 Pt195
Cisplatin (cis-Diammineplatinum(II) dichloride) Merck P4394
Clear 1.5 mL tube Fisher Scientific 11926955
Collagenase, Type II Worthington Biochemical Corporation LS004177
Counting chamber Merck BR718620-1EA
CXCR4/SDF1 (Clone: 2B11/CXCR4 ) BD Biosciences 551852 Gd158; In-house conjugated
DAPI (1 mg/mL) BD Biosciences 564907
Dark 1.5 mL tube Fisher Scientific 15386548
Dispase II Thermo Fisher Scientific 17105041
Dissection Scissors Fine Science Tools 14568-09
DMEM (low glucose, with pyruvate) Thermo Fisher Scientific 11885-092
EDTA (Ethylenediaminetetraacetic acid disodium salt) Merck E5134 Na2EDTA-2H20
EQ Four Element Calibration Beads (EQ beads) Standard Biotools 201078 Calibration beads
Fetal Bovine Serum, qualified, Brazil origin Thermo Fisher Scientific 10270106
Forceps Dumont #5SF Fine Science Tools 11252-00
Forceps Dumont #7 Hounisen.com 1606.3350
Goat serum Thermo Fisher Scientific 16210-072
Helios CyTOF system Standard Biotools N/A
Horse Serum, heat inactivated, New Zealand origin Thermo Fisher Scientific 26-050-088
IdU Merck I7125 I127
Iridium-Intercalator Standard Biotools 201240 Ir191/193
Isoflurane/Attane Vet ScanVet Vnr 055226
Methanol Fisher Scientific M/3900/17
Myf5 (Clone: C-20) Santa Cruz Biotechnology Sc-302 Yb173; In-house conjugated
MyoD (Clone: 5.8A) BD Biosciences 554130 Dy164; In-house conjugated
MyoG (Clone: F5D) BD Biosciences 556358 Gd160; In-house conjugated
Nalgene Rapid-Flow Sterile Disposable Bottle Top 0.20 μM PES Filters Thermo Fisher Scientific 595-4520
Notexin Latoxan L8104 Resuspend to 50 µg/ml in sterile PBS. Keep stocks (e.g. 50 µl) at -20 °C
Nutrient mixture F-10 (Ham's) Thermo Fisher Scientific 31550031
pAkt (Clone: D9E) Standard Biotools 3152005A Sm152
Pax7 (Clone: PAX7) Santa Cruz Biotechnology Sc-81648 Eu153; In-house conjugated
Penicillin-Streptomycin (10,000 U/mL) (Pen/Strep) Thermo Fisher Scientific 15140122
PES Filter Units 0.20 μM Fisher Scientific 15913307
PES Syringe Filter Fisher Scientific 15206869
Petri dish Sarstedt 82.1472.001
PFA 16% EM grade MP Biomedicals 219998320
Potassium chloride (KCl) Fisher Scientific 10375810
Potassium phosphate, monobasic, anhydrous (KH2PO4) Fisher Scientific 10573181
pRb (Clone: J112-906) Standard Biotools 3166011A Er166
pS6 kinase (Clone: N7-548) Standard Biotools 3172008A Yb172
Sca-1 (Clone: E13-161.7) BD Biosciences 553333 Nd142; In-house conjugated
Sodium Azide Sigma Aldrich S2002
Sodium chloride (NaCl) Fisher Scientific 10553515
Sodium phosphate, dibasic, heptahydrate (Na2HPO4-6H2O) Merck S9390
Sterile saline solution 0.9% Fresenius B306414/02
α7 integrin (Clone: 3C12) MBL international K0046-3 Ho165; In-house conjugated
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References

  1. Mukund, K., Subramaniam, S. Skeletal muscle: A review of molecular structure and function in health and disease. Wiley Interdiscip Rev Syst Biol Med. 12 (1), 1462 (2020).
  2. Feige, P., Brun, C. E., Ritso, M., Rudnicki, M. A. Orienting muscle stem cells for regeneration in homeostasis, aging, and disease. Cell Stem Cell. 23 (5), 653-664 (2018).
  3. Mauro, A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol. 9 (2), 493-495 (1961).
  4. Seale, P., et al. Pax7 is required for the specification of myogenic satellite cells. Cell. 102 (6), 777-786 (2000).
  5. Fuchs, E., Blau, H. M. Tissue stem cells: Architects of their niches. Cell Stem Cell. 27 (4), 532-556 (2020).
  6. Hernández-hernández, J. M., et al. The myogenic regulatory factors, determinants of muscle development, cell identity and regeneration. Semin Cell Dev Biol. 72, 10-18 (2017).
  7. Zammit, P. S. Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF4 in skeletal muscle, satellite cells and regenerative myogenesis. Semin Cell Dev Biol. 72, 19-32 (2017).
  8. Sabourin, L. A., Rudnicki, M. A. The molecular regulation of myogenesis. Clin Genet. 57 (1), 16-25 (2000).
  9. Cooper, R. N., et al. In vivo satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle. J Cell Sci. 112 (17), 2895-2901 (1999).
  10. Rudnicki, M. A., Jaenisch, R. The MyoD family of transcription factors and skeletal myogenesis. Bioessays. 17 (3), 203-209 (1995).
  11. Braun, T., Arnold, H. H. Inactivation of Myf-6 and Myf-5 genes in mice leads to alterations in skeletal muscle development. EMBO J. 14 (6), 1176-1186 (1995).
  12. Yablonka-Reuveni, Z. Development and postnatal regulation of adult myoblasts. Microsc Res Tech. 30 (5), 366-380 (1995).
  13. Braun, T., et al. MyoD expression marks the onset of skeletal myogenesis in Myf-5 mutant mice. Development. 120 (11), 3083-3092 (1994).
  14. Rudnicki, M. A., et al. MyoD or Myf-5 is required for the formation of skeletal muscle. Cell. 75 (7), 1351-1359 (1993).
  15. Montarras, D., et al. Developmental biology: Direct isolation of satellite cells for skeletal muscle regeneration. Science. 309 (5743), 2064-2067 (2005).
  16. Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. Self-renewal and expansion of single transplanted muscle stem cells. Nature. 456 (7221), 502-506 (2008).
  17. Cerletti, M., et al. Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles. Cell. 134 (1), 37-47 (2008).
  18. Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat Protoc. 10 (10), 1612-1624 (2015).
  19. Porpiglia, E., et al. High-resolution myogenic lineage mapping by single-cell mass cytometry. Nat Cell Biol. 19 (5), 558-567 (2017).
  20. Behbehani, G. K., Bendall, S. C., Clutter, M. R., Fantl, W. J., Nolan, G. P. Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry Part A. 81 (7), 552-566 (2012).
  21. Hartmann, F. J., et al. . Mass Cytometry: Methods and Protocols. , (2019).
  22. Devine, R. D., Behbehani, G. K. Use of the pyrimidine analog, 5-iodo-2′-deoxyuridine (IdU) with cell cycle markers to establish cell cycle phases in a mass cytometry platform. J Vis Exp. (176), e60556 (2021).
  23. Bendall, S. C., et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science. 332 (6030), 687-696 (2011).
  24. Nag, A. C., Foster, J. D. Myogenesis in adult mammalian skeletal muscle in vitro. J Anat. 132, 1-18 (1981).
  25. Le Moigne, A., et al. Characterization of myogenesis from adult satellite cells cultured in vitro). Int J Dev Biol. 34, 171-180 (1990).
  26. Yablonka-Reuveni, Z. Development and postnatal regulation of adult myoblasts. Microsc Res Tech. 30 (5), 366-380 (1995).
  27. Chu, C., Cogswell, J., Kohtz, D. S. MyoD functions as a transcriptional repressor in proliferating myoblasts. J Biol Chem. 272 (6), 3145-3148 (1997).
  28. Shah, B., Hyde-Dunn, J., Jones, G. E. Proliferation of murine myoblasts as measured by bromodeoxyuridine incorporation. Methods in Mol Biol. 75, 349-355 (1997).
  29. Springer, M. L., Blau, H. M. High-efficiency retroviral infection of primary myoblasts. Somat Cell Mol Genet. 23 (3), 203-209 (1997).
  30. Rando, T. A., Blau, H. M. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol. 125 (6), 1275-1287 (1994).
  31. Springer, M. L., Rando, T. A., Blau, H. M. Gene delivery to muscle. Curr Protoc Hum Genet. , (2002).
  32. Cull-Candy, S. G., Fohlman, J., Gustavsson, D., Lullmann-Rauch, R., Thesleff, S. The effects of taipoxin and notexin on the function and fine structure of the murine neuromuscular junction. Neuroscience. 1 (3), 175-180 (1976).
  33. Francis, B., John, T. R., Seebart, C., Kaiser, New toxins from the venom of the common tiger snake (Notechis scutatus scutatus). Toxicon. 29 (1), 85-96 (1991).
  34. Gonzalez, V. D., et al. High-grade serous ovarian tumor cells modulate NK cell function to create an immune-tolerant microenvironment. Cell Rep. 36 (9), 109632 (2021).
  35. Delgado-Gonzalez, A., et al. Measuring trogocytosis between ovarian tumor and natural killer cells. STAR Protoc. 3 (2), 101425 (2022).
  36. Finck, R., et al. Normalization of mass cytometry data with bead standards. Cytometry Part A. 83 (5), 483-494 (2013).
  37. Leipold, M. D., Maecker, H. T. Mass cytometry: protocol for daily tuning and running cell samples on a CyTOF mass cytometer. J Vis Exp. (69), e4398 (2012).
  38. McCarthy, R. L., Duncan, A. D., Barton, M. C. Sample preparation for mass cytometry analysis. J Vis Exp. (122), e54394 (2017).
  39. Kotecha, N., Krutzik, P. O., Irish, J. M. Web-based analysis and publication of flow cytometry experiments. Curr Protoc Cytom. , (2010).
  40. Fienberg, H. G., Simonds, E. F., Fantl, W. J., Nolan, G. P., Bodenmiller, B. A platinum-based covalent viability reagent for single-cell mass cytometry. Cytometry Part A. 81 (6), 467-475 (2012).
  41. Kimball, A. K., et al. A beginner’s guide to analyzing and visualizing mass cytometry data. J Immunol. 200 (1), 3-22 (2018).
  42. Weber, L. M., Robinson, M. D. Comparison of clustering methods for high-dimensional single-cell flow and mass cytometry data. Cytometry Part A. 89 (12), 1084-1096 (2016).
  43. Samusik, N., Good, Z., Spitzer, M. H., Davis, K. L., Nolan, G. P. Automated mapping of phenotype space with single-cell data. Nat Methods. 13 (6), 493-496 (2016).
  44. Ornatsky, O. I., et al. Study of cell antigens and intracellular DNA by identification of element-containing labels and metallointercalators using inductively coupled plasma mass spectrometry. Anal Chem. 80 (7), 2539-2547 (2008).
  45. Relaix, F., et al. Perspectives on skeletal muscle stem cells. Nat Commun. 12 (1), 692 (2021).
  46. de Morree, A., et al. Staufen1 inhibits MyoD translation to actively maintain muscle stem cell quiescence. Proc Natl Acad Sci U S A. 114 (43), E8996-E9005 (2017).
  47. Luo, D., et al. Deltex2 represses MyoD expression and inhibits myogenic differentiation by acting as a negative regulator of Jmjd1c. Proc Natl Acad Sci U S A. 114 (15), E3071-E3080 (2017).
  48. Wersto, R. P., et al. Doublet discrimination in DNA cell-cycle analysis. Cytometry. 46 (5), 296-306 (2001).
  49. Porpiglia, E., Blau, H. M. Plasticity of muscle stem cells in homeostasis and aging. Curr Opin Genet Dev. 77, 101999 (2022).
  50. Porpiglia, E., et al. Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration. Cell Stem Cell. 29 (12), 1653-1668 (2022).
  51. Brunet, A., Goodell, M. A., Rando, T. A. Ageing and rejuvenation of tissue stem cells and their niches. Nat Rev Mol Cell Biol. 24 (1), 45-62 (2022).
  52. Danielli, S. G., et al. Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance. Sci Adv. 9 (6), (2023).
  53. de Morree, A., Rando, T. A. Regulation of adult stem cell quiescence and its functions in the maintenance of tissue integrity. Nat Rev Mol Cell Biol. 24 (5), 334-354 (2023).
  54. Yucel, N., et al. Glucose metabolism drives histone acetylation landscape transitions that dictate muscle stem cell glucose metabolism drives histone acetylation landscape transitions that dictate muscle stem cell function. Cell Rep. 27 (13), 3939-3955 (2019).
  55. Tierney, M. T., Sacco, A. Inducing and evaluating skeletal muscle injury by notexin and barium chloride. Methods Mol Biol. 1460, 53-60 (2016).
  56. Hardy, D., et al. Comparative study of injury models for studying muscle regeneration in mice. PLoS One. 11 (1), 0147198 (2016).
  57. Call, J. A., Lowe, D. A. Eccentric contraction-induced muscle injury: Reproducible, quantitative, physiological models to impair skeletal muscle’s capacity to generate force. Methods Mol Biol. 1460, 3-18 (2016).
  58. Garry, G. A., Antony, M. L., Garry, D. J. Cardiotoxin Induced Injury and Skeletal Muscle Regeneration. Methods Mol Biol. 1460, 61-71 (2016).
  59. Le, G., Lowe, D. A., Kyba, M. Freeze injury of the tibialis anterior muscle. Methods Mol Biol. 1460, 33-41 (2016).
  60. Borok, M., et al. Progressive and coordinated mobilization of the skeletal muscle niche throughout tissue repair revealed by single-cell proteomic analysis. Cells. 10 (4), 744 (2021).
  61. Petrilli, L. L., et al. High-dimensional single-cell quantitative profiling of skeletal muscle cell population dynamics during regeneration. Cells. 9 (7), 1723 (2020).
  62. Giordani, L., et al. High-dimensional single-cell cartography reveals novel skeletal muscle-resident cell populations. Mol Cell. 74 (3), 609-621 (2019).
  63. Hartmann, F. J., et al. Scalable conjugation and characterization of immunoglobulins with stable mass isotope reporters for single-cell mass cytometry analysis. Methods Mol Biol. 1989, 55-81 (2019).
  64. Frimand, Z., Das Barman, S., Kjær, T. R., Porpiglia, E., de Morrée, A. Isolation of quiescent stem cell populations from individual skeletal muscles. J Vis Exp. (190), e64557 (2022).
  65. Krutzik, P. O., Nolan, G. P. Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A. 55 (2), 61-70 (2003).
  66. Bodenmiller, B., et al. Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators. Nat Biotechnol. 30 (9), 858-867 (2012).
  67. Schulz, K. R., Danna, E. A., Krutzik, P. O., Nolan, G. P. Single-cell phospho-protein analysis by flow cytometry. Curr Protoc Immunol. , 11-20 (2012).
  68. Krutzik, P. O., Clutter, M. R., Nolan, G. P. Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry. J Immunol. 175 (4), 2357-2365 (2005).
  69. Krutzik, P. O., Irish, J. M., Nolan, G. P., Perez, O. D. Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin Immunol. 110 (3), 206-221 (2004).
  70. Han, G., Spitzer, M. H., Bendall, S. C., Fantl, W. J., Nolan, G. P. Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry. Nat Protoc. 13 (10), 2121-2148 (2018).
  71. Chevrier, S., et al. Compensation of signal spillover in suspension and imaging mass cytometry. Cell Syst. 6 (5), 612-620 (2018).
  72. Bjornson, Z. B., Nolan, G. P., Fantl, W. J. Single-cell mass cytometry for analysis of immune system functional states. Curr Opin Immunol. 25 (4), 484-494 (2013).
  73. Kalina, T., Lundsten, K., Engel, P. Relevance of antibody validation for flow cytometry. Cytometry A. 97 (2), 126-136 (2020).
  74. Baumgarth, N., Roederer, M. A practical approach to multicolor flow cytometry for immunophenotyping. J Immunol Methods. 243 (1-2), 77-97 (2000).
  75. Roederer, M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry. 45 (3), 194-205 (2001).
  76. Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. A., Herzenberg, L. A. New approaches to fluorescence compensation and visualization of FACS data. Clin Immunol. 110 (3), 277-283 (2004).
  77. Cossarizza, A., et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition). Eur J Immunol. 51 (12), 2708-3145 (2021).

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Keywords: Skeletal Muscle RegenerationMuscle Stem CellsMuscle ProgenitorsSingle-cell Mass CytometryCyTOFFACSMuscle InjuryMyogenesisCell Surface MarkersTranscription Factors
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Andersen, S. H., Kjær, T. R., Ghimire, K., Porpiglia, E. Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry. J. Vis. Exp. (202), e65944, doi:10.3791/65944 (2023).
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