Measuring Cellular Mechanotransduction Response

Mammalian cells expressing GFPs were trypsinized and loaded into the MPA observation chamber, which was filled with Leibovitz L-15 media at room temperature (Life Technologies). A pressure difference was generated by adjusting the height of a motor-driven water manometer. Cells were aspirated with a fixed pressure of 0.15 nN/µm² for NIH 3T3 cells and 0.2 nN/µm² for HeLa cells, due to the unique cortical tensions of these cells in an attempt to produce similar levels of cell deformation (Schiffhauer et al., 2016 (link)). All cells which demonstrated separation of cell membrane from the cortex at any time during recording were discarded. Images were collected every 10 s for 5 min with an Olympus IX81 microscope using a UPanFL 40× (1.30 NA) oil objective and an Andor iXON EMCCD camera. Acquisition was performed using Metamorph software and were analyzed using Image J (National Institutes of Health). After background correction, the fluorescence intensity (mean gray value) at the accumulation sites inside the micropipette were normalized against the opposite cortex of the cell (I_p/I_o). Measurements taken at the peak of mechanoresponse were then normalized against the initial I_p/I_o value to correct for any initial variation in cortical uniformity.