Analysis and quantification of in vitro myoblast fusion using the LADD Multiple Stain

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.