Handling and Assessment of Human Primary Prostate Organoid Culture
| Autor: | Larisa Nonn, Tara McCray, Gail S. Prins, Zachary Richards, Joseph Marsili |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Male
General Chemical Engineering Biology Immunofluorescence Article General Biochemistry Genetics and Molecular Biology Flow cytometry Tissue Culture Techniques 03 medical and health sciences 0302 clinical medicine Prostate medicine Organoid Humans 030304 developmental biology Whole mount 0303 health sciences General Immunology and Microbiology medicine.diagnostic_test General Neuroscience Epithelial Cells Protein subcellular localization prediction Cell biology Staining Organoids medicine.anatomical_structure Antibody staining 030217 neurology & neurosurgery |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area. DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers. |
| Databáze: | OpenAIRE |
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