Figure 2.

Method for culturing of C2C12 cells to obtain high yields of AChR clusters. (a) Examples of suboptimal (red boxes) and optimal (green box) cell densities for passaging. Yellow arrows show the area of excessive cell contacts that should be avoided. Cells were plated at the same time at varying densities. Cells highlighted by the green box were seeded at approximately 1.0× 10⁶ cells/cm² cultured on a 10 cm dish. (b) Schematic diagram of C2C12 cell passages to obtain a high number of cell stocks at an equally low number of passages. (c) Schematic diagram of the key steps during C2C12 cells culturing that are important for obtaining high yields of AChR clusters. The images show optimal (green box) and suboptimal (red boxes) cell densities for fusion initiation. Red arrowheads show areas that are not covered by cells. Yellow arrows show regions where single-cell morphology can be observed. For the highest amount of fully differentiated myotubes with AChR clusters, myoblasts should form a dense cell layer in which cell morphology is difficult to distinguish. (d) Schematic diagram of the key steps during human primary myoblast culturing that are important for obtaining high yields of AChR clusters. The differences in the protocol for handling of C2C12 cells and human primary myoblasts are highlighted in blue. Scale bar = 600 μm in a and 450 μm in c.