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 …
Divulgaciones
The authors have nothing to disclose.
Materials
Mice
Lgr5-EGFP-IRES-creERT2 (Lgr5-GFP)
The Jackson Laboratory
Growth culture medium
Advanced DMEM F12 (DMEM/F12)
ThermoFisher
12634010
B-27 Supplement, minus vitamin A
ThermoFisher
12587010
Stock Concentration: 50x
GlutaMAX (glutamine)
ThermoFisher
35050038
Stock Concentration: 100x
Hepes
ThermoFisher
15630056
Stock Concentration: 1 M
Murine EGF
R&D
2028-EG-200
Stock Concentration: 500 µg/mL in PBS
murine Noggin
R&D
1967-NG/CF
Stock Concentration: 100 µg/mL in PBS
Murine R-spondin1
R&D
3474-RS-050
Stock Concentration: 50 µg/mL in PBS
N-2 Supplement
ThermoFisher
17502048
Stock Concentration: 100x
Penicillin-Streptomycin (P/S)
ThermoFisher
15140122
Stock Concentration: 100x (10,000 units/mL of penicillin and 10,000 µg/mL of streptomycin)
Flow Cytometry Assay to Quantify Oxidative Stress: An Assay to Quantify Reactive Oxygen Species in Intestinal Organoids Using Fluorogenic Probes. J. Vis. Exp. (Pending Publication), e21221, doi: (2023).