11/12/2023 0 Comments Dead cells logo transparentThen, cell nuclei were stained with Hoechst 33342 overnight at 4 ☌, and spheroids were transparentized with ScaleS.Īcquisition and analysis of fluorescent imagesĬonfocal fluorescent images of the above-mentioned spheroids were obtained using the FV3000 laser confocal microscope. After incubation for 1 day, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% TritonX-100/PBS. Staurosporine (STS) at various concentrations and NucView550 were added to each well 8 days after the start of cell culture. The study demonstrates that drug-independent and drug-dependent cell death can be confirmed using a clearing reagent and NoviSight™ software.Ī cell suspension of HT-29 was seeded into PrimeSurface®96U plates (SUMITOMO BAKELITE CO., LTD), a U bottom well plate, at 500 cells/well. In this study, confocal images of fluorescent stained cancer spheroids, treated with a drug, fixed, and made transparent, were subjected to 3D quantitative analysis using NoviSight™ software. However, the development of tissue clearing technology now makes it possible to observe inside 3D samples while preserving their structural integrity. Previously, each section had to be separately prepared to observe the inside of 3D samples. A spheroid model is expected to recapitulate tumor microenvironment. The distance from the surface of a tumor to the core has a diffusion gradient of oxygen or nutrients, and this severe environment causes resting cells or dead cells deep inside the tumor tissue. The ratio of dead cells inside spheroids and drug efficacy can be quantitatively evaluated using a confocal microscope and NoviSight™ software's cell counting module.
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