Primary ovarian cancer cells (NACT8) were cultured in a low-attachment 6-well plate for 96 hours. The culture was treated with 10 μM JSI-124 or DMSO for 30 or 120 min. Spheroids were isolated by passing the suspension through a 20 μm filter. Reverse filtration was performed to collect spheroids larger than 20 μm into a 6-well plate. Cells were washed and spheroids were collected for the mesothelial clearance assay. A mesothelial cells monolayer was prepared by plating mesothelial cells on glass-bottom dishes (Mat-TEK Corporation) coated with 5 μg/mL of fibronectin (Sigma, USA). Cells were maintained in culture until confluent (~48 hours after plating). Suspended NACT8 cell spheroids were collected and added to a confluent mesothelial monolayer expressing green fluorescent protein (GFP), allowed to attach for 30–60 min, and imaged for up to 16 hours using a Nikon Ti-E Inverted Motorized Widefield Fluorescence Microscope equipped with incubation chamber. Only spheres that remained attached during the experiment were used for quantification. Mesothelial clearance was quantified as previously described44 ,45 .
Ti e inverted motorized widefield fluorescence microscope
Manufactured by Nikon
Sourced in United States
The Ti-E Inverted Motorized Widefield Fluorescence Microscope is a laboratory equipment designed for optical microscopy. It features a motorized, inverted optical system with widefield fluorescence capabilities.
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2 protocols using ti e inverted motorized widefield fluorescence microscope
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Mesothelial Clearance Assay for Ovarian Cancer
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Mesothelial Clearance Assay for Ovarian Cancer
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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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