Hit stage top incubation system

Live-cell, wide-field phase microscopy videos were acquired on a Nikon Eclipse Ti, equipped with a Nikon 20× Plan 0.40 NA air objective and a DS-Qi2 complementary metal-oxide semiconductor camera. Images were acquired every 1 min. Cells in DMEM supplemented with 10 mM Hepes, were placed in a Tokai Hit stage-top incubation system during imaging at 37°C and 5% CO₂, and an orange, glass filter was placed on top of the condenser to restrict short wavelengths of light. Rates of ingression were calculated using ImageJ. Images were first aligned using the “StackReg” plugin, and a line of width 3 was drawn across the division plane of the dividing cells. Kymographs were generated using the “MultipleKymograph” plugin in ImageJ. Rates of ingression were then manually calculated from when the cell started to ingress until ingression had stopped, resulting in distance over time measurements.

Images were acquired with a Hamamatsu ORCA-Fusion Gen III sCMOS camera on a Nikon Eclipse Ti2E, an Andor cooled CCD camera on a Nikon Eclipse Ti, or an Andor CSU-W1 two-camera spinning disk module, an Andor Zyla sCMOS camera, and an Andor ILE laser module on a Nikon Eclipse Ti. Objectives included Nikons CFI Plan Apo Lambda 60×, 1.4 numerical aperture (NA) oil immersion (OI); CFI Super Plan Fluor LWD 20× AMD, 0.7 NA air; Plan Apo VC 60×, 1.4 NA OI; and Plan Apo Lambda 60×, 1.4 NA OI. Z-step size was either 0.2, 0.5, or 1 μm. For quantification of proteins, all images were acquired with the same acquisition parameters and exposure times. Additional details are in the supplemental information.
Live-cell imaging was performed in modified rose chambers (RPE-1 H2B-GFP) or on 35 mm glass-bottom dishes (MatTek #P35G-1.5-14-C) coated with Matrigel or Laminin-521 (hPSCs) at 37°C in a humidified environment with 5% CO₂ (Tokai Hit Stage-top Incubation System) using the microscopes described above with binning set to 2 × 2. Additional details are in the supplemental information.