A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The overall goal of this procedure is to determine forces on the linker of the nucleoskeleton cytoskeleton, or LINC complex, through FRET microscopy in living cells via a biosensor designed for the LINC complex protein, nesprin-2G. This method can help answer keys questions in the field of mechanotransduction, such as the forces placed on proteins in the nucleus of living cells, and how these forces change under variant conditions. The main advantage of this technique is its ability to measure very small levels of force on proteins and living cells.

Generally, individuals new to this method will struggle with capturing images. Optimizing the microscope laser power and gain to receive a good signal between all experimental groups is difficult. To begin, grow NIH 3T3 fiber blast cells to between 70%and 90%confluence in a six-well cell culture dish in a standard cell culture incubator with temperature and CO2 regulation.