Fluorescent characterization of differentiated myotubes using flow cytometry
Flow cytometry is routinely used in the assessment of skeletal muscle progenitor cell (myoblast) populations. However, a full gating strategy, inclusive of difficult to interpret forward and side scatter data, which documents cytometric analysis of differentiated myoblasts (myotubes) has not been re...
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| Veröffentlicht in: | Cytometry. Part A 2024-05, Vol.105 (5), p.332-344 |
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| creator | Nolan, Andy Heaton, Robert A. Adamova, Petra Cole, Paige Turton, Nadia Gillham, Scott H. Owens, Daniel J. Sexton, Darren W. |
| description | Flow cytometry is routinely used in the assessment of skeletal muscle progenitor cell (myoblast) populations. However, a full gating strategy, inclusive of difficult to interpret forward and side scatter data, which documents cytometric analysis of differentiated myoblasts (myotubes) has not been reported. Beyond changes in size and shape, there are substantial metabolic and protein changes in myotubes allowing for their potential identification within heterogenous cell suspensions. To establish the utility of flow cytometry for determination of myoblasts and myotubes, C2C12 murine cell populations were assessed for cell morphology and metabolic reprogramming. Laser scatter, both forward (FSC; size) and side (SSC; granularity), measured cell morphology, while mitochondrial mass, reactive oxygen species (ROS) generation and DNA content were quantified using the fluorescent probes, MitoTracker green, CM‐H2DCFDA and Vybrant DyeCycle, respectively. Immunophenotyping for myosin heavy chain (MyHC) was utilized to confirm myotube differentiation. Cellular viability was determined using Annexin V/propidium iodide dual labelling. Fluorescent microscopy was employed to visualize fluorescence and morphology. Myotube and myoblast populations were resolvable through non‐intuitive interpretation of laser scatter‐based morphology assessment and mitochondrial mass and activity assessment. Myotubes appeared to have similar sizes to the myoblasts based on laser scatter but exhibited greater mitochondrial mass (159%, p |
| doi_str_mv | 10.1002/cyto.a.24822 |
| format | Article |
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However, a full gating strategy, inclusive of difficult to interpret forward and side scatter data, which documents cytometric analysis of differentiated myoblasts (myotubes) has not been reported. Beyond changes in size and shape, there are substantial metabolic and protein changes in myotubes allowing for their potential identification within heterogenous cell suspensions. To establish the utility of flow cytometry for determination of myoblasts and myotubes, C2C12 murine cell populations were assessed for cell morphology and metabolic reprogramming. Laser scatter, both forward (FSC; size) and side (SSC; granularity), measured cell morphology, while mitochondrial mass, reactive oxygen species (ROS) generation and DNA content were quantified using the fluorescent probes, MitoTracker green, CM‐H2DCFDA and Vybrant DyeCycle, respectively. Immunophenotyping for myosin heavy chain (MyHC) was utilized to confirm myotube differentiation. Cellular viability was determined using Annexin V/propidium iodide dual labelling. Fluorescent microscopy was employed to visualize fluorescence and morphology. Myotube and myoblast populations were resolvable through non‐intuitive interpretation of laser scatter‐based morphology assessment and mitochondrial mass and activity assessment. Myotubes appeared to have similar sizes to the myoblasts based on laser scatter but exhibited greater mitochondrial mass (159%, p < 0.0001), ROS production (303%, p < 0.0001), DNA content (18%, p < 0.001) and expression of MyHC (147%, p < 0.001) compared to myoblasts. Myotube sub‐populations contained a larger viable cluster of cells which were unable to be fractionated from myoblast populations and a smaller population cluster which likely contains apoptotic bodies. Imaging of differentiated myoblasts that had transited through the flow cytometer revealed the presence of intact, ‘rolled‐up’ myotubes, which would alter laser scatter properties and potential transit through the laser beam. Our results indicate that myotubes can be analyzed successfully using flow cytometry. Increased mitochondrial mass, ROS and DNA content are key features that correlate with MyHC expression but due to myotubes ‘rolling up’ during flow cytometric analysis, laser scatter determination of size is not positively correlated; a phenomenon observed with some size determination particles and related to surface properties of said particles. We also note a greater heterogeneity of myotubes compared to myoblasts as evidenced by the 2 distinct sub‐populations. We suggest that acoustic focussing may prove effective in identifying myotube sub populations compared to traditional hydrodynamic focussing.</description><identifier>ISSN: 1552-4922</identifier><identifier>EISSN: 1552-4930</identifier><identifier>DOI: 10.1002/cyto.a.24822</identifier><identifier>PMID: 38092660</identifier><language>eng</language><publisher>Hoboken, USA: John Wiley & Sons, Inc</publisher><subject>Annexin V ; Apoptosis ; C2C12 ; Cell morphology ; Cell suspensions ; Cell viability ; Cells (biology) ; Clusters ; Cytology ; Deoxyribonucleic acid ; DNA ; DNA probes ; Flow ; Flow cytometry ; Fluorescence ; Fluorescent indicators ; Heterogeneity ; Iodides ; Labeling ; Laser beams ; Lasers ; Metabolism ; Microscopy ; Mitochondria ; Mitochondrial DNA ; Morphology ; Myoblasts ; Myosin ; Myotubes ; Populations ; Progenitor cells ; Propidium iodide ; Reactive oxygen species ; Scattering ; Size determination ; Skeletal muscle ; Surface properties</subject><ispartof>Cytometry. 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Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c3592-69e8dec5844e0c2823a03c83e9658f090633a5285f81421b71e2d750f95bd8203</cites><orcidid>0000-0001-5514-8414 ; 0000-0003-3344-3150</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fcyto.a.24822$$EPDF$$P50$$Gwiley$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fcyto.a.24822$$EHTML$$P50$$Gwiley$$Hfree_for_read</linktohtml><link.rule.ids>314,776,780,1411,27901,27902,45550,45551</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/38092660$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nolan, Andy</creatorcontrib><creatorcontrib>Heaton, Robert A.</creatorcontrib><creatorcontrib>Adamova, Petra</creatorcontrib><creatorcontrib>Cole, Paige</creatorcontrib><creatorcontrib>Turton, Nadia</creatorcontrib><creatorcontrib>Gillham, Scott H.</creatorcontrib><creatorcontrib>Owens, Daniel J.</creatorcontrib><creatorcontrib>Sexton, Darren W.</creatorcontrib><title>Fluorescent characterization of differentiated myotubes using flow cytometry</title><title>Cytometry. Part A</title><addtitle>Cytometry A</addtitle><description>Flow cytometry is routinely used in the assessment of skeletal muscle progenitor cell (myoblast) populations. However, a full gating strategy, inclusive of difficult to interpret forward and side scatter data, which documents cytometric analysis of differentiated myoblasts (myotubes) has not been reported. Beyond changes in size and shape, there are substantial metabolic and protein changes in myotubes allowing for their potential identification within heterogenous cell suspensions. To establish the utility of flow cytometry for determination of myoblasts and myotubes, C2C12 murine cell populations were assessed for cell morphology and metabolic reprogramming. Laser scatter, both forward (FSC; size) and side (SSC; granularity), measured cell morphology, while mitochondrial mass, reactive oxygen species (ROS) generation and DNA content were quantified using the fluorescent probes, MitoTracker green, CM‐H2DCFDA and Vybrant DyeCycle, respectively. Immunophenotyping for myosin heavy chain (MyHC) was utilized to confirm myotube differentiation. Cellular viability was determined using Annexin V/propidium iodide dual labelling. Fluorescent microscopy was employed to visualize fluorescence and morphology. Myotube and myoblast populations were resolvable through non‐intuitive interpretation of laser scatter‐based morphology assessment and mitochondrial mass and activity assessment. Myotubes appeared to have similar sizes to the myoblasts based on laser scatter but exhibited greater mitochondrial mass (159%, p < 0.0001), ROS production (303%, p < 0.0001), DNA content (18%, p < 0.001) and expression of MyHC (147%, p < 0.001) compared to myoblasts. Myotube sub‐populations contained a larger viable cluster of cells which were unable to be fractionated from myoblast populations and a smaller population cluster which likely contains apoptotic bodies. Imaging of differentiated myoblasts that had transited through the flow cytometer revealed the presence of intact, ‘rolled‐up’ myotubes, which would alter laser scatter properties and potential transit through the laser beam. Our results indicate that myotubes can be analyzed successfully using flow cytometry. Increased mitochondrial mass, ROS and DNA content are key features that correlate with MyHC expression but due to myotubes ‘rolling up’ during flow cytometric analysis, laser scatter determination of size is not positively correlated; a phenomenon observed with some size determination particles and related to surface properties of said particles. We also note a greater heterogeneity of myotubes compared to myoblasts as evidenced by the 2 distinct sub‐populations. We suggest that acoustic focussing may prove effective in identifying myotube sub populations compared to traditional hydrodynamic focussing.</description><subject>Annexin V</subject><subject>Apoptosis</subject><subject>C2C12</subject><subject>Cell morphology</subject><subject>Cell suspensions</subject><subject>Cell viability</subject><subject>Cells (biology)</subject><subject>Clusters</subject><subject>Cytology</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA probes</subject><subject>Flow</subject><subject>Flow cytometry</subject><subject>Fluorescence</subject><subject>Fluorescent indicators</subject><subject>Heterogeneity</subject><subject>Iodides</subject><subject>Labeling</subject><subject>Laser beams</subject><subject>Lasers</subject><subject>Metabolism</subject><subject>Microscopy</subject><subject>Mitochondria</subject><subject>Mitochondrial DNA</subject><subject>Morphology</subject><subject>Myoblasts</subject><subject>Myosin</subject><subject>Myotubes</subject><subject>Populations</subject><subject>Progenitor cells</subject><subject>Propidium iodide</subject><subject>Reactive oxygen species</subject><subject>Scattering</subject><subject>Size determination</subject><subject>Skeletal muscle</subject><subject>Surface properties</subject><issn>1552-4922</issn><issn>1552-4930</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><sourceid>24P</sourceid><recordid>eNp9kE1LxDAQhoMoft88S8GLB3edTJpucpTFL1jwogdPIZtOtNI2mrRI_fV2XfXgwdMMzMPDOy9jRxymHADP3dCFqZ1irhA32C6XEie5FrD5uyPusL2UXgCEBIHbbEco0FgUsMsWV3UfIiVHbZe5Zxut6yhWH7arQpsFn5WV9xTHa2U7KrNmCF2_pJT1qWqfMl-H92yVoKEuDgdsy9s60eH33GcPV5f385vJ4u76dn6xmDghNU4KTaokJ1WeEzhUKCwIpwTpQioPGgohrEQlveI58uWME5YzCV7LZakQxD47XXtfY3jrKXWmqcYP6tq2FPpkUAPqGVczMaInf9CX0Md2TGcEyDGNzvlKeLamXAwpRfLmNVaNjYPhYFYtm9WPxpqvlkf8-FvaLxsqf-GfWkcgXwPvVU3DvzIzf7y_u1h7PwE8E4ib</recordid><startdate>202405</startdate><enddate>202405</enddate><creator>Nolan, Andy</creator><creator>Heaton, Robert A.</creator><creator>Adamova, Petra</creator><creator>Cole, Paige</creator><creator>Turton, Nadia</creator><creator>Gillham, Scott H.</creator><creator>Owens, Daniel J.</creator><creator>Sexton, Darren W.</creator><general>John Wiley & Sons, Inc</general><general>Wiley Subscription Services, Inc</general><scope>24P</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>7TK</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0001-5514-8414</orcidid><orcidid>https://orcid.org/0000-0003-3344-3150</orcidid></search><sort><creationdate>202405</creationdate><title>Fluorescent characterization of differentiated myotubes using flow cytometry</title><author>Nolan, Andy ; 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Part A</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nolan, Andy</au><au>Heaton, Robert A.</au><au>Adamova, Petra</au><au>Cole, Paige</au><au>Turton, Nadia</au><au>Gillham, Scott H.</au><au>Owens, Daniel J.</au><au>Sexton, Darren W.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Fluorescent characterization of differentiated myotubes using flow cytometry</atitle><jtitle>Cytometry. Part A</jtitle><addtitle>Cytometry A</addtitle><date>2024-05</date><risdate>2024</risdate><volume>105</volume><issue>5</issue><spage>332</spage><epage>344</epage><pages>332-344</pages><issn>1552-4922</issn><eissn>1552-4930</eissn><abstract>Flow cytometry is routinely used in the assessment of skeletal muscle progenitor cell (myoblast) populations. However, a full gating strategy, inclusive of difficult to interpret forward and side scatter data, which documents cytometric analysis of differentiated myoblasts (myotubes) has not been reported. Beyond changes in size and shape, there are substantial metabolic and protein changes in myotubes allowing for their potential identification within heterogenous cell suspensions. To establish the utility of flow cytometry for determination of myoblasts and myotubes, C2C12 murine cell populations were assessed for cell morphology and metabolic reprogramming. Laser scatter, both forward (FSC; size) and side (SSC; granularity), measured cell morphology, while mitochondrial mass, reactive oxygen species (ROS) generation and DNA content were quantified using the fluorescent probes, MitoTracker green, CM‐H2DCFDA and Vybrant DyeCycle, respectively. Immunophenotyping for myosin heavy chain (MyHC) was utilized to confirm myotube differentiation. Cellular viability was determined using Annexin V/propidium iodide dual labelling. Fluorescent microscopy was employed to visualize fluorescence and morphology. Myotube and myoblast populations were resolvable through non‐intuitive interpretation of laser scatter‐based morphology assessment and mitochondrial mass and activity assessment. Myotubes appeared to have similar sizes to the myoblasts based on laser scatter but exhibited greater mitochondrial mass (159%, p < 0.0001), ROS production (303%, p < 0.0001), DNA content (18%, p < 0.001) and expression of MyHC (147%, p < 0.001) compared to myoblasts. Myotube sub‐populations contained a larger viable cluster of cells which were unable to be fractionated from myoblast populations and a smaller population cluster which likely contains apoptotic bodies. Imaging of differentiated myoblasts that had transited through the flow cytometer revealed the presence of intact, ‘rolled‐up’ myotubes, which would alter laser scatter properties and potential transit through the laser beam. Our results indicate that myotubes can be analyzed successfully using flow cytometry. Increased mitochondrial mass, ROS and DNA content are key features that correlate with MyHC expression but due to myotubes ‘rolling up’ during flow cytometric analysis, laser scatter determination of size is not positively correlated; a phenomenon observed with some size determination particles and related to surface properties of said particles. We also note a greater heterogeneity of myotubes compared to myoblasts as evidenced by the 2 distinct sub‐populations. We suggest that acoustic focussing may prove effective in identifying myotube sub populations compared to traditional hydrodynamic focussing.</abstract><cop>Hoboken, USA</cop><pub>John Wiley & Sons, Inc</pub><pmid>38092660</pmid><doi>10.1002/cyto.a.24822</doi><tpages>13</tpages><orcidid>https://orcid.org/0000-0001-5514-8414</orcidid><orcidid>https://orcid.org/0000-0003-3344-3150</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Annexin V Apoptosis C2C12 Cell morphology Cell suspensions Cell viability Cells (biology) Clusters Cytology Deoxyribonucleic acid DNA DNA probes Flow Flow cytometry Fluorescence Fluorescent indicators Heterogeneity Iodides Labeling Laser beams Lasers Metabolism Microscopy Mitochondria Mitochondrial DNA Morphology Myoblasts Myosin Myotubes Populations Progenitor cells Propidium iodide Reactive oxygen species Scattering Size determination Skeletal muscle Surface properties |
| title | Fluorescent characterization of differentiated myotubes using flow cytometry |
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