eoe sub articles

EoE Sub Articles

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
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	<li>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 &#181;g/mL of murine Noggin 500 ng/mL of mouse R-Spondin1 (see&#160;Table of Mater...

flow cytometry assay to quantify oxidative stress: an assay to quantify reactive oxygen species in intestinal organoids using fluorogenic probes

Source: Stedman, A., et al., <a href="https://app.jove.com/v/62880">Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe.</a>&#160;J. Vis. Exp. (2021).
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.

<img alt="Figure 1" src="/files/ftp_upload/21221/21221_Figure1.jpg" /> 
Figure 1: Representative images of crypts and organoids.&#160;(A)&#160;Example of fraction F1 obtained after the first incubation with EDTA, enriched in villi (square), with some debris (star) and crypts (circle). (B)&#160;Example of fraction F4 enriched in crypts. (C)&#160;Suspension presenting only isolated crypts obtained after the filtration ...

Flow Cytometry Assay to Quantify Oxidative Stress: An Assay to Quantify Reactive Oxygen Species in Intestinal Organoids Using Fluorogenic Probes

Source: Stedman, A., et al., Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe.&#160;J. ...

Intestinal cells produce reactive oxygen species - ROS. Moderate ROS levels maintain cellular homeostasis, while high ROS levels can cause cell death.
To quantify ROS levels in vitro, begin with a culture of mouse intestinal 3D organoids. These organoids exhibit gut-epithelium architecture having crypt-like protrusions harboring green fluorescence-expressing stem cells. They also contain villus-like differentiated cells at the periphery, surrounding the central lumen comprising dead cells and debris. All these cells produce different levels of ROS.
Now, treat the organoids in the designated wells with ROS modulators. Based on their nature, modulators can act as ROS inducers or inhibitors. After incubating for the required duration, remove the spent medium.
Add a proteolytic enzyme to the culture, disrupting the organoids&#39; 3D architecture and releasing different cells in suspension. Next, add a ROS-sensitive fluorogenic probe and incubate.
Upon entering the cells, the probes react with ROS and get oxidized to produce red fluorescence. Treat the cells with DAPI - a DNA-binding fluorescent dye - which selectively stains the nuclei of dead cells - creating a contrast with the live cells.
Using a flow cytometer, analyze the DAPI-negative live cells to obtain a red fluorescence signal corresponding to their ROS level. Depending on the nature of modulators, inducers increase ROS levels while the inhibitors decrease them.

Flow Cytometry Assay to Quantify Oxidative Stress: An Assay to Quantify Reactive Oxygen Species in Intestinal Organoids Using Fluorogenic Probes

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