Bsi scmos camera

Brightfield images of adherently cultured and DOX-induced OV90, and ACI23 cells were taken on a Nikon Eclipse Ts2 inverted microscope with NIS elements software (Nikon, Melville, NY) at 10 × and 20 × magnification. For immunofluorescent staining, OV90 cells were plated on glass coverslips and cultured for 3 days in DOX-containing media. Cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100, blocked with 10% goat serum in PBS for 1 h at room temperature, and then incubated with primary antibodies (1:500) in 10% goat serum in PBS overnight in a humidified chamber, at 4 °C. The following day, coverslips were washed three times with PBS, incubated with fluorescent secondary antibodies (A-11034, A-21236, Thermo Scientific, Waltham, MA) at 1:2000, for 1 h at room temperature protected from light, washed three times with PBS and mounted onto glass slides with Fluoroshield with DAPI (F6057, Millipore Sigma, Burlington, MA). Images were taken on an inverted Nikon Ti2-E microscope (Nikon, Melville, NY), equipped with a Yokogawa SoRa CSU-W1 spinning disk unit (Yokogawa, Sugar Land, TX) and a BSI sCMOS camera (Teledyne Photometrics, Tuscon, AZ) at 60 × magnification. Signal intensity was measured using NIS Elements AR software (Nikon, Melville, NY).

Sphere formation of OV90, OVCAR8 and OVCAR3 cells was performed as previously described (15 (link)). Cells were seeded at 2,000 cells/well in 96-well ULA plates (3474, Corning, Corning, NY), in TEM with indicated drugs for 7 days, fresh culture medium containing growth factors and the drugs was replenished every 48 h. After 7 days the spheres were incubated with DRAQ5 (Thermo Fisher Scientific, Waltham, MA, USA) at 1 µM for 15 min prior to imaging using an inverted Nikon Ti2-E microscope (Nikon, Melville, NY), equipped with a Yokogawa SoRa CSU-W1 spinning disk unit (Yokogawa, Sugar Land, TX) and a BSI sCMOS camera (Teledyne Photometrics, Tuscon, AZ). Images of spheroids were acquired using the automated acquisition module (JOBS) to image each well using a 10× plan-apochromat N.A. 0.45 objective lens, 200 ms exposure. Quantification of spheroids was performed using NIS Elements software version 5.30 (Nikon, Melville, NY). Images were processed using Segment.ai trained on human-recognized, hand-traced spheroids. Segment.ai learned how to trace spheroids in subsequent images. The number of spheroids measuring an area of >1,000 μm² were counted.