Eclipse ti e microscope system

CELLview^TM 4 compartment glass bottom dishes (VWR International, Radnor, PA, US) were pre-coated with fibronectin/gelatin for 1 h at 37 °C. Primary fibroblasts were seeded at a density of 2.5 × 10³ and allowed to attach overnight at 37 °C in the presence of 5% CO₂. Cells received fresh BIO-AMF^TM-2 medium supplemented with 10 mM HEPES and dishes were mounted onto an inverted Nikon Eclipse Ti-E microscope system equipped with a 37 °C incubator (Nikon). Phase contrast images were acquired at 10 min intervals for 8 h with a 10×/0.30NA objective. For analysis, cells were manually tracked with the MTrackJ plugin in ImageJ. Cells were excluded from the analysis if they collided with another cell or underwent division during the imaging period. The Dicty Tracking 1.4 software package was used to calculate cell trajectories, random migration speeds, directionality ratios and the mean square displacement of cells⁵⁶ .

Phase contrast images were taken of the monolayer for a duration of 6–12 h using a Nikon Eclipse Ti-E microscope system. The majority of the data was taken using a 10x air objective (CFI Plan Fluor DLL, 10x, N.A. 0.30, W.D. 16.0 mm, Ph1, Nikon), acquiring an image every 2 min. To increase spatial and temporal resolution, imaging was repeated with a 20x objective (CFI Plan Fluor DLL, 20x, N.A. 0.50, W.D. 2.1 mm, Ph1, Nikon) taking an image every 0.5 min (see Supplementary Methods for discussion on this).
Dividing cells with a distance of at least 150 μm from other dividing cells during the duration of the observation period were identified manually in the phase contrast images. The dividing cell was centered in the 300 × 300 μm² frame and the frame was rotated so that the daughter cells would move along a horizontal axis away from the site of division (Fig. 1 and Supplementary Fig. S1). The image sequence spanned from +/−40 min from the site of cell division.

Human keratinocytes were cultured to 70% confluence on glass coverslips and then fixed on ice in either methanol for 2 min or 4% paraformaldehyde for 10 min followed by 0.2% Triton X-100 for 7 min. Primary antibodies described above were detected with Alexa Fluor-conjugated secondary antibodies. Wide field fluorescence microscopy was performed using a DMRXA2 microscope (Leica, Wetzler, Germany) equipped with a 63×/1.32 NA oil immersion objective and narrow band pass filters. Images were acquired with an ORCA digital camera (Hamamatsu Photonics, Bridgewater, NJ) and processed using Simple PCI software (Hamamatsu Corporation, Sewickley, PA). Super-resolution microscopy was performed using a Nikon N-SIM system on an Eclipse Ti-E microscope system equipped with a 100×/1.49 NA oil immersion objective, 488- and 561-nm solid-state lasers in 3D structured illumination microscopy mode. Images were captured using an EM charge-coupled device camera (DU-897, Andor Technology) and reconstructed using NIS-Elements software with the N-SIM module (version 3.22, Nikon). Colocalization analysis was performed by obtaining Mander’s coefficient using ImageJ plugin JACoP [35] (link).