A simple trypsin resistance assay for muscle and other cell fusion

Muscle cells fusing in vitro have long provided biologists with a tool to study development and gene expression. However, many such studies used morphological assays of cell fusion. We present here a method for assaying fusion at a specific, operationally defined step. Muscle cells grown in monolayer are exposed to trypsin-EDTA solution at 37 degrees C; the trypsin is inactivated, the cells fixed in Lugol's iodine, and 200 to 300 nuclei are counted as being single or multiple. The presence of EDTA is important under standard conditions for muscle culture; however, little difference is seen in divalent cation-depleted cultures. Therefore, for consistency EDTA can be included in all assays. Samples are stable for over 24 hr, with no cell loss from trypsinization or fixation. This assay exploits a specific stage of muscle fusion, trypsin-resistant contact, to provide a rapid, simple, and observer-independent assay for an early state of muscle fusion. The assay can be used to measure fusion between any nucleated cells.