MCs (3 × 104) were cultured on a 24-well plate in different experimental conditions. Serum-free medium alone or with ET-1 and/or BQ123+BQ788 or conditioned media (CM) from SKOV3 or SKOV3 treated with AMB or from HEY or HEY treated with AMB was added and left for different times (24, 48, 72 h) at 37°C. Total cells from each well were then recuperated by using Trypsin-EDTA 1X in PBS 100 ML (Euroclone) solution and counted by using an automated cell counter (Beckman Coulter). The experiment was performed in triplicates for all conditions described and repeated at least three times. As indicated, we used Cyto3DTM Live-Dead Assay Kit (TheWell Bioscience, Inc., North Brunswick, NJ, United States) to determine the live/dead nucleated cells by using a dual-fluorescence system of Acridine orange (AO) and propidium iodide (PI), both nuclear staining (nucleic acid binding) dyes. All live nucleated cells fluoresce green, and all dead nucleated cells fluoresce red. Several images were taken by using Bio-Rad ZOE fluorescent cell image under a phase-contrast microscope (Bio-Rad Laboratories).
Phase contrast microscope
Manufactured by Bio-Rad
Sourced in United States
The Phase-contrast microscope is a type of optical microscope that uses phase-contrast illumination to produce high-contrast images of transparent samples, such as living cells, without the need for staining. The core function of this microscope is to enhance the visibility of structures within the sample by converting variations in the refractive index into variations in brightness in the final image.
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Lab products found in correlation
3 protocols using phase contrast microscope
1
Effect of ET-1 and AMB on MCs
2
Spheroid Live-Dead Assay Protocol
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Cells (1 × 103) were cultured in complete medium in a U-shaped bottom 96-well plate and centrifuged at 200 g for 5 min. 24 h after spheroid formation, serum-free medium, as indicated, was added and left for 72 h at 37 °C. After 72 h, the Cyto3DTM Live–Dead Assay Kit (TheWell Bioscience, Inc., North Brunswick, NJ, United States) was used to determine the live/dead nucleated cells using a dual-fluorescence system of acridine orange (AO) and propidium iodide (PI), both nuclear staining (nucleic acid binding) dyes. All live nucleated cells fluoresce green, and all dead nucleated cells fluoresce red. Several images were obtained using a Bio-Rad ZOE fluorescent cell imager under a phase-contrast microscope (Bio-Rad Laboratories). Image analysis was performed using the FIJI software by calculating the mean gray value of the green and red channels separately, and then the green/red ratio was calculated.
3
Cell Viability and Morphology Observation
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For the observation of cell viability, morphology, and confluence, phase-contrast microscope was used (Zoe, BioRad, CA, USA).
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