title: Analysis and quantification of in vitro myoblast fusion using the LADD Multiple Stain.
abstract: Myoblast fusion, which is essential for muscle development, regeneration, and repair, can be assessed in vitro via the calculation of a fusion index. Traditionally, this requires use of either immunocytochemistry or fluorescently-labeled cytoskeletal staining, followed by microscopy and laborious analysis. The expense and time-consuming nature of the optimization and application of antibody-based techniques such as immunocytochemistry, as well as the need for specialized analytical equipment such as fluorescence microscopes, presents a barrier to the routine analysis of this crucial step during terminal differentiation. Here, we describe (i) a novel use of the commonly available LADD Multiple Stain for visualization of myoblast fusion in vitro; (ii) the optimization of a simple image analysis method to generate quick, quantifiable data representative of a fusion index; and (iii) the use of a protocol combining these two procedures to investigate in vitro myoblast fusion in a simple and efficient manner as proof-of-concept.
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, South Africa.
authors.forename: Rhys
authors.initials: R
authors.lastname: McColl
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, South Africa.
authors.forename: Mthokozisi
authors.initials: M
authors.lastname: Nkosi
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, South Africa.
authors.forename: Celia
authors.initials: C
authors.lastname: Snyman
authors.affiliation: Department of Biochemistry, School of Life Sciences, University of KwaZulu-Natal, South Africa.
authors.forename: Carola
authors.initials: C
authors.lastname: Niesler
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002459:Cell Fusion; D002460:Cell Line; D004396:Coloring Agents / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008853:Microscopy / Q000379:methods*; D032446:Myoblasts / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D012394:Rosaniline Dyes / Q000737:chemistry / Q000378:metabolism; D014048:Tolonium Chloride / Q000737:chemistry / Q000378:metabolism
chemical_list: D004396:Coloring Agents; D012394:Rosaniline Dyes; D014048:Tolonium Chloride
keywords: LADD; differentiation; fusion; microscopy; myoblast; skeletal muscle