Quantifying ICAM-1+ Myoblast Fusion

Fusion competent ICAM-1+ myoblasts were fluorescently labeled using CellTracker™ Green CMFDA (Life Technologies; 2.5 µM); whereas, fusion competent EV myoblasts were not fluorescently labeled. Cells were collected using StemPro® Accutase® (Invitrogen), suspended in differentiation medium (200 cells/µl), and ICAM-1+ and EV myoblasts were mixed in equal number in polypropylene tubes. Tubes were placed in a shaking water bath for 2 h and aliquots of cells were immobilized to slides using a cytospin centrifuge^{4 (link)}. Cells were fixed in 4% formaldehyde, stained with WGA (Alexa Fluor® 350; Thermo Scientific), and mounted with Fluoromount-G^TM (SouthernBiotech). Images in six fields of view were captured using a 10X objective on an epifluorescence microscope.
The number of aggregates and the number of myoblasts within an aggregate were quantified using a macro function written for Image Pro 7 (Media Cybernetics). The number of ICAM-1+ cells within an aggregate was expressed as a percentage of the total number of WGA +cells within the aggregate. The number of EV cells was calculated by subtracting the total number of WGA+ cells from the number of ICAM-1+ cells within an aggregate. On average, ~2,000 myoblasts per slide were counted in 6 independent experiments.