Sweptfield eclipse te2000 e confocal microscope

1×10⁵ cells in full medium were plated on top of 4-5mg/ml Matrigel-coated delta-T dishes (Bioptechs). After 1 hour, the medium was removed and the cell layer was covered with more Matrigel. After polymerizing for 30 minutes at 37°C, media was re-added. Live-cell imaging was conducted every 10 minutes for 24 hours at 37°C via Nikon Sweptfield/Eclipse/TE2000-E confocal microscope (20X objective). Time lapse images were imported into ImageJ software and combined to form video files. Quantification methods are detailed in the Supplemental Materials and Methods.

Cells treated with si/sgRNAs against NEDD9 or RhoA and/or overexpressing CA-RhoA and/or treated with 50μM Y-27632 or 100nM MLN8237 for 24h were re-suspended in 1.5mg/ml collagen and placed into 3D-Chemotaxis μ-slides (Ibidi) according to manufacturer protocol. Fetal bovine serum was added to the left media reservoir to create a chemoattractant gradient. When used, Y-27632 and/or MLN8237 were also added to both reservoirs to keep the drugs present during the assay. Cells were imaged every 15 minutes via DIC 10X objective on a Nikon Sweptfield/Eclipse/TE2000-E confocal microscope for 23 hours. Time lapse images were imported into ImageJ software and combined to form video files. Quantification methods are detailed in the Supplemental Materials and Methods.

Cells were pretreated overnight with 50μM Y-27632 or 100nM MLN8237, and 3×10⁴ cells were plated in 8-well chamber slides (ThermoFisher) coated with 150μl 4mg/ml Matrigel. After 1 hour, the medium was removed and the cells covered with another 200μl Matrigel, followed by full media after Matrigel polymerization. Cells were live imaged in 5μm Z-steps away from the cell seeding position at 72 hours using a Nikon Sweptfield/Eclipse/TE2000-E confocal microscope. Invasion was quantified by total cell fluorescence intensity at the furthest distance cells reached (100μm). 3D invasion boxes were constructed using the Volume-Viewer plugin for ImageJ to show cell fluorescence signal throughout the Matrigel from the seeding position.