20 na 0.8 air objective

Microfluidic migration devices with precisely defined constrictions were prepared as described previously^{25 (link),46 (link)}. Devices were coated with 50 μg/mL of type-I rat tail collagen (BD Biosciences) in 0.02N acetic acid overnight at 4°C. Approximately 80,000 cells were seeded (in DMEM supplemented with 10% FBS and 1% PenStrep) per migration chamber. Devices were placed in a tissue-culture incubator (37°C) for 5–6 hours to allow the cells to adhere. Subsequently, media was changed to phenol-red free Leibovitz L15 media supplemented with 10% FBS and 1% PenStrep before mounting the device on an inverted microscope (Zeiss Observer Z1) equipped with temperature-controlled stage (37°C) for live-cell imaging. The media reservoirs of the device were covered with glass coverslips to minimize evaporation during live-cell imaging. Cells were imaged for 14–16 hours at 10 minute intervals with a CCD camera (Photometrics CoolSNAP KINO) using a Zeiss 20′/NA 0.8 air objective. Acquired image sequences were analyzed for nuclear rupture frequency, duration, and transit time of cells through 1×5μm², 2×5μm², and 15×5μm² constrictions using Zen (Zeiss) software and a custom-written MATLAB 2016a script for automated image analysis.