Mesothelial-clearance assays were performed as described previously9 (link) with minor alterations. The mesothelial cells were incubated at 37 °C for 16 h to form confluent monolayers. After the 16-h incubations, OVCAR3 and KURAMOCHI spheroids (100 cells per spheroid) were transferred to the wells containing the mesothelial monolayers.
Imaging was performed as described previously9 (link) using a Nikon Ti-E Inverted Motorized Widefield Fluorescence Microscope (Nikon, Melville, NY, USA). Over 20 spheroids were imaged per condition. Phase-contrast and GFP images were captured at 0 and 24 h. The non-fluorescent surface area created by the invading spheroid in the GFP mesothelial monolayer images was measured at 24 h and divided by the initial two-dimensional area of the cancer spheroid at the initial seeding time (time 0 or 0.5 h). Twenty biological replicates were performed to calculate P-values for each experiment.
Imaging was performed as described previously9 (link) using a Nikon Ti-E Inverted Motorized Widefield Fluorescence Microscope (Nikon, Melville, NY, USA). Over 20 spheroids were imaged per condition. Phase-contrast and GFP images were captured at 0 and 24 h. The non-fluorescent surface area created by the invading spheroid in the GFP mesothelial monolayer images was measured at 24 h and divided by the initial two-dimensional area of the cancer spheroid at the initial seeding time (time 0 or 0.5 h). Twenty biological replicates were performed to calculate P-values for each experiment.
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