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Flow Cytometry Assay to Quantify Oxidative Stress: An Assay to Quantify Reactive Oxygen Species in Intestinal Organoids Using Fluorogenic Probes
Overview
This video demonstrates a flow cytometry assay to quantify the ROS levels in 3D intestinal organoids obtained from GFP-expressing transgenic mice. This method provides an in vitro experimental model to investigate the efficacy of test compounds on ROS production.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of reagents and materials for culturing intestinal organoids
- To prepare growth culture medium, mix advanced DMEM/F-12 supplemented with 1x glutamine, 1x penicillin/streptomycin (P/S) solution, 10 mM of HEPES, 50 ng/mL of murine EGF, 20 µg/mL of murine Noggin 500 ng/mL of mouse R-Spondin1 (see Table of Mater.......
Representative Results
Figure 1: Representative images of crypts and organoids. (A) Example of fraction F1 obtained after the first incubation with EDTA, enriched in villi (square), with some debris (star) and crypts (circle). (B) Example of fraction F4 enriched in crypts. (C) Suspension presenting only isolated crypts obtained after the filtration .......
This article has been published
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Source: Stedman, A., et al., Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe. J. Vis. Exp. (2021).
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