Journal: STAR Protocols

Article Title: Protocol for serial organoid formation assay using primary colorectal cancer tissues to evaluate cancer stem cell activity

doi: 10.1016/j.xpro.2022.101218

Figure Lengend Snippet: Examples of organoid detection methods using high content imaging (A) 3D-embedded organoids can be detected, counted, and scored by high content imaging using ImageXpress Automated Cell Imaging Systems, or equivalent platforms. (B) Example of organoid detection based on a size exclusion threshold, where cellular structures below a definite size are not counted from brightfield images. A color mask is applied by the analysis software over each structure considered as an organoid. Scale bar = 70 μm. (C) Example for the use of a fluorescent (GFP) reporter system outlining a specific population of HCT116 organoids to be counted in an experiment. A color mask is applied by the analysis software over each structure presenting a fluorescence signal above a definite background threshold (FLUOR Mask). Scale bar = 70 μm. (D) Identification of live organoid structures using the cell permeable, fluorescent dye Calcein AM (green). While several live organoids are detectable in DMSO-treated wells, only residual live cells are observed upon treatments with the anticancer small molecule YB-0158. Scale bar = 70 μm.

Article Snippet: YB-0158 , MedChemExpress , Cat# HY-136541.

Techniques: Imaging, Software, Fluorescence

Journal: STAR Protocols

Article Title: Protocol for serial organoid formation assay using primary colorectal cancer tissues to evaluate cancer stem cell activity

doi: 10.1016/j.xpro.2022.101218

Figure Lengend Snippet: Schematic analysis of the colorectal tumor organoids resulting from serial passaging (A) Brightfield images of secondary organoids depicting size difference over the growth stages. Scale bar = 70 μm. (B) High content imaging can be used to establish organoid counts, size, and shape (form factor: FF) variations for control vs. treated conditions. (C) Secondary organoid counts obtained from the serial passage of primary organoid cultures treated with 3 compounds targeting CSC functions in colorectal tumors (BIX01294, UNC0642: G9a inhibitor. YB-0158: Sam68 modulator). Organoid counts were established based on size exclusion (< 40 μm). n≥4 biological replicates from 3 independent patients; ∗∗: p≤0.0053, ∗∗∗: p≤0.0001). (D) Fluorescence imaging of a colorectal tumor organoid by confocal microscopy. A composite image of proliferative cells (Ki-67; green), adherens junctions (E-Cadherin; red), and nuclei (Hoechst 33342; blue) is presented. Scale bar = 30 μm. (E) Tridimensional reconstruction of CRC patient-derived spheroids and organoids from confocal fluorescence microscopy images using the Imaris 9.5 rendering platform. For each type of multicellular structure, immunostaining of the proliferation marker Ki-67 and intestinal self-renewal marker Bmi1 are presented. Scale bar = 30 μm.

Article Snippet: YB-0158 , MedChemExpress , Cat# HY-136541.

Techniques: Passaging, Imaging, Fluorescence, Confocal Microscopy, Derivative Assay, Microscopy, Immunostaining, Marker