Fluorescent Probe Imaging Assay: A Technique to Visualize Oxidative Stress in the Reactive Oxygen Species Inducer-Treated Cultured Intestinal Organoid Cells
Published: April 30, 2023
Abstract
Source: Stedman, A. et al., Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe. J. Vis. Exp. (2021).
This video describes the technique of visualizing oxidative stress in cultured organoids. This work helps in understanding the level of oxidative stress due to ROS accumulation in the various cell types present in an organoid after treatment with different ROS modulators.
Protocol
1. Visualization of oxidative stress in 3D organoids by confocal microscopy Take the organoids plated in the µ-Slide 8 well chambers and add 1 µL N-acetyl cysteine or NAC stock solution in the corresponding wells to obtain a final concentration of 1 mM. Incubate for 1 h at 37 °C and 5% CO2. Add 1 µL tBHP stock solution in the corresponding wells to obtain a final concentration of 200 µM. Incubate for 30 min at 37 °C and 5% CO2.<…
Disclosures
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 | |
| Penicillin-Streptomycin (P/S) | ThermoFisher | 15140122 | Stock Concentration: 100x (10,000 units/mL of penicillin and 10,000 µg/ mL of streptomycin) |
| FLuoroBrite DMEM (DMEM no phenol red) | ThermoFisher | A1896701 | |
| Hoechst 33342 | ThermoFisher | H3570 | stock at 10 mg/mL |
| Matrigel Growth Factor Reduced, Phenol Red Free (Basement Membrane Matrix) | Corning | 356231 | once received thaw o/n in the fridge, keep for 1h on ice and, make 500 mL aliquots and store at -20 °C |
| µ-Slide 8 Well chambers | Ibidi | 80826 | |
| N-acetylcysteine (NAC) | Sigma | A9165 | |
| tert-Butyl hydroperoxide (tBCHP)solution (70%wt. In H2O2) | Sigma | 458139 | |
| Programs and Equipment | |||
| Fiji/ImageJ | https://imagej.net/software/fiji/ downloads images generation | ||
| Observer.Z1 | Zeiss | confocal system | |
| Opterra (swept-field confocal) | Bruker | confocal system | |
| high speed EMCCD Camera Evolve Delta 512 | Photometrics | confocal system | |
| Prism | GraphPad Software | statistical analysis |
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Cite This Article
Fluorescent Probe Imaging Assay: A Technique to Visualize Oxidative Stress in the Reactive Oxygen Species Inducer-Treated Cultured Intestinal Organoid Cells. J. Vis. Exp. (Pending Publication), e21220, doi: (2023).