Using small intestinal epithelial cells, the organoid culture was performed as described previously 33 (link). Briefly, the organoids were cultured in matrigel with advanced DMEM/F12 medium (Invitrogen) supplemented with 50 ng/mL EGF (Invitrogen), R-Spondin1 conditioned medium (R&D Systems), and 100 ng/mL Noggin (Peprotech). The culture was passaged once, and the crypt length was measured under a dissecting microscope. Organoid cell proliferation was detected using the Click-iT EdU Imaging System (Invitrogen).
Click it edu imaging system
Manufactured by Thermo Fisher Scientific
The Click-iT EdU Imaging System is a fluorescence-based cell proliferation assay. It allows for the detection and quantification of DNA synthesis in proliferating cells. The system utilizes the incorporation of a thymidine analog, EdU (5-ethynyl-2'-deoxyuridine), into DNA during active DNA synthesis. The incorporated EdU can then be detected using a fluorescent azide through a copper-catalyzed click reaction.
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2 protocols using click it edu imaging system
1
Intestinal Epithelial Organoid Culture
2
Organoid Cultivation from Intestinal Cells
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The cultivation of organoids from small intestinal epithelial cells was conducted following previously established protocols [25 (link)]. In summary, the resulting organoids were maintained within Matrigel, employing advanced DMEM/F12 medium (HY-K3002; Sigma) that was supplemented with 50 ng/mL epidermal growth factor (EGF) (HY-P70590AF; Sigma), R-Spondin1 conditioned medium (R&D Systems, Minneapolis, MN, USA), and 100 ng/mL Noggin (250-38-1MG; PeproTech, Cranbury, NJ, USA). The cultures underwent a single passage, with crypt length measurements performed under a dissecting microscope. The proliferation of organoid cells was assessed using the Click-iT EdU Imaging System (Invitrogen).
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