Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2010 Sep 1;588(Pt 17):3307-20.
doi: 10.1113/jphysiol.2010.190876. Epub 2010 Jul 12.

Satellite cell number and cell cycle kinetics in response to acute myotrauma in humans: immunohistochemistry versus flow cytometry

Bryon R McKay  1 , Kyle G Toth, Mark A Tarnopolsky, Gianni Parise
Affiliations
Comparative Study

Satellite cell number and cell cycle kinetics in response to acute myotrauma in humans: immunohistochemistry versus flow cytometry

Bryon R McKay et al. J Physiol. .

Abstract

In humans, muscle satellite cell (SC) enumeration is an important measurement used to determine the myogenic response to various stimuli. To date, the standard practice for enumeration is immunohistochemistry (IHC) using antibodies against common SC markers (Pax7, NCAM). Flow cytometry (FC) analysis may provide a more rapid and quantitative determination of changes in the SC pool with potential for additional analysis not easily achievable with standard IHC. In this study, FC analysis revealed that the number of Pax7(+) cells per milligram isolated from 50 mg of fresh tissue increased 36% 24 h after exercise-induced muscle injury (300 unilateral maximal eccentric contractions). IHC analysis of Pax7 and neural cell adhesion molecule (NCAM) appeared to sufficiently and similarly represent the expansion of SCs after injury (28-36% increase). IHC and FC data illustrated that Pax7 was the most widely expressed SC marker in muscle cross-sections and represented the majority of positive cells, while NCAM was expressed to a lesser degree. Moreover, FC and IHC demonstrated a similar percentage change 24 h after injury (36% increase, Pax7; 28% increase, NCAM). FC analysis of isolated SCs revealed that the number of Pax7(+) cells per milligram in G(2)/M phase of the cell cycle increased 202% 24 h after injury. Number of cells per milligram in G(0)/G(1) and cells in S-phase increased 32% and 59% respectively. Here we illustrate the use of FC as a method for enumerating SC number on a per milligram tissue basis, providing a more easily understandable relation to muscle mass (vs. percentage of myonuclei or per myofibre). Although IHC is a powerful tool for SC analysis, FC is a fast, reliable and effective method for SC quantification as well as a more informative method for cell cycle kinetics of the SC population in humans.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Satellite cell enumeration via flow cytometry before (PRE) and 24 h after muscle damage
A, total mononuclear cells isolated from whole muscle biopsies. B, total Pax7+ cells mg−1 isolated from whole muscle biopsies. Pax7+ cells mg−1 increased 36% (P= 0.04; mean ±s.d., n= 12). C, representative flow cytometry histograms of total FITC (Pax7) fluorescence versus cell number of a secondary only control (CON), a pre-exercise (PRE) and 24 h post-exercise (24 h) cell sample. D, representative flow cytometry histograms of forward scatter (FS, y-axis) vs. side scatter (SS, x-axis) of an ungated sample and Pax7+ cells (gated on Pax7 and propidium iodide PRE and 24 h).
Figure 3
Figure 3. Immunohistochemical analysis of the satellite cell response to muscle damage before (PRE) and 24 h after muscle damage
A, representative image of Pax7/laminin co-immunofluorescent stain. Arrow denotes a Pax7+ nucleus (red) beneath the basal lamina (green). Scale bar = 50 μm. B, representative image of a C-Met/laminin co-immunofluorescent stain. Arrow denotes a C-Met+ nucleus (green) beneath the basal lamina (red). Scale bar = 50 μm. C, representative image of NCAM/laminin co-immunofluorescent stain. Arrow denotes a NCAM+ cell (red) beneath the basal lamina (green). Scale bar = 50 μm. D, total number of myonuclei per muscle. E, SC number (per 100 myofibres) by SC marker. *Main effect of time (P < 0.05). F, SC number (as a percentage of total myonuclei) by SC marker. *Significant effect of time between Pax7 and C-met(P < 0.05). †Significant difference between Pax7 and C-met positive cells at PRE (P < 0.05). All data are means ±s.d., n= 12.
Figure 2
Figure 2. Cell cycle analysis via flow cytometry
A and B, representative DNA histograms of SC (Pax7+ cells) isolated from muscle biopsies before (A, PRE) and 24 h after (B, 24 h) muscle damage. Cells in Go/G1 of the cell cycle are depicted in blue, cells with increasing DNA content (S-phase) are depicted in purple and cells with 2 compliments of DNA (cells in G2/M) are depicted in red. C, total number of Pax7+ cells mg−1 which are in Go/G1 of the cell cycle before (PRE) and 24 h after muscle damage. D, total number of Pax7+ cells mg−1 in S-phase of the cell cycle before (PRE) and 24 h after muscle damage (24 h). E, total number of Pax7+ cells mg−1 in G2/M of the cell cycle before (PRE) and 24 h after muscle damage (24 h). F, total number of Pax7+ cells mg−1 in each phase of the cell cycle as a proportion of the total number of SCs before (PRE) and 24 h after muscle damage. *Significant difference from PRE (P < 0.05). All data are means ±s.d., n= 12.
Figure 4
Figure 4. Co-labelling of satellite cell markers before (PRE) and 24 h after muscle damage
A, co-localization of C-Met and Pax7 with DAPI, scale bar = 50 μm; higher magnification of box: (i) C-Met (red), (ii) nucleus (DAPI, blue), (iii) Pax7 (green), and (iv) merged image showing co-localization. B, co-localization of NCAM and Pax7 with DAPI, scale bar = 50 μm; higher magnification of box: (i) NCAM (red), (ii) nucleus (DAPI, blue), (iii) Pax7 (green), and (iv) merged image showing co-localization. C, co-positive SC number by marker as a percentage of total myonuclei. *Significant difference between NCAM/Pax7 and C-Met/Pax7 at PRE (P < 0.05). †Significant effect of time for C-Met/Pax7 (P < 0.05). D, single positive cells (cells expressing only one SC marker) as a percentage of total myonuclei. *Significant difference at PRE between Pax7 only vs. C-Met and NCAM only (P < 0.05). E, representative flow cytometry histogram of muscle derived mononuclear cells stained for NCAM and Pax7. Cells expressing both NCAM and Pax7 (E2) represent approximately 95% of all positive cells, while NCAM only cells (E1) and Pax7 only (E4) represent approximately 5% of all positive cells. Cells negative for both SC markers are shown in quadrant E3. F, comparison of flow cytometry and IHC enumeration techniques: comparison of the percentage change in SC number 24 h after muscle damage analysed by flow cytometry and IHC for Pax7 and NCAM. *Significant effect of time (PRE vs. 24 h: P < 0.05). All data presented as means ±s.d., n= 12.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alexander CM, Puchalski J, Klos KS, Badders N, Ailles L, Kim CF, Dirks P, Smalley MJ. Separating stem cells by flow cytometry: reducing variability for solid tissues. Cell Stem Cell. 2009;5:579–583. - PMC - PubMed
    1. Bar H, Strelkov SV, Sjoberg G, Aebi U, Herrmann H. The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation? J Struct Biol. 2004;148:137–152. - PubMed
    1. Baroffio A, Bochaton-Piallat ML, Gabbiani G, Bader CR. Heterogeneity in the progeny of single human muscle satellite cells. Differentiation. 1995;59:259–268. - PubMed
    1. Beaton LJ, Allan DA, Tarnopolsky MA, Tiidus PM, Phillips SM. Contraction-induced muscle damage is unaffected by vitamin E supplementation. Med Sci Sports Exerc. 2002a;34:798–805. - PubMed
    1. Beaton LJ, Tarnopolsky MA, Phillips SM. Contraction-induced muscle damage in humans following calcium channel blocker administration. J Physiol. 2002b;544:849–859. - PMC - PubMed

Publication types