Name: analyzing oxidative stress in murine intestinal organoids using reactive oxygen species-sensitive fluorogenic probe
Uploaded: 2021-09-17T14:45:00
Description: Watch this Scientific Journal Video about Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe at JoVE.com

This work was supported by French National Research Agency (ANR) grant 17-CE14-0022 (i-Stress).

All animal experiments were carried out after approval by the Institut Pasteur Use Committee and by the French Ministry of Agriculture no. 2016-0022. All the steps are performed inside a tissue culture hood.
1. Preparation of reagents and materials for culturing intestinal organoids
<ol>
	<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...

<ol>
	<li>Aviello, G., Knaus, U. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidases+and+ROS+signaling+in+the+gastrointestinal+tract+review-article.">NADPH oxidases and ROS signaling in the gastrointestinal tract review-article.</a> Mucosal Immunology. 11 (4), 1011-1023 (2018).</li><li>Holmstr&#246;m, K. M., Finkel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+and+physiological+consequences+of+redox-dependent+signalling.">Cellular mechanisms and physiological consequences of redox-dependent signalling.</a> Nature Reviews Molecular Cell Biology. 15 (6), 411-421 (2014).</li><li>vander Post, S., Birchenough, G. M. H., Held, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOX1-dependent+redox+signaling+potentiates+colonic+stem+cell+proliferation+to+adapt+to+the+intestinal+microbiota+by+linking+EGFR+and+TLR+activation.">NOX1-dependent redox signaling potentiates colonic stem cell proliferation to adapt to the intestinal microbiota by linking EGFR and TLR activation.</a> Cell Reports. 35 (1), 108949 (2021).</li><li>Schieber, M., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+function+in+redox+signaling+and+oxidative+stress.">ROS function in redox signaling and oxidative stress.</a> Current Biology. 24 (10), 453-462 (2014).</li><li>Myant, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+production+and+NF-&#954;B+activation+triggered+by+RAC1+facilitate+WNT-driven+intestinal+stem+cell+proliferation+and+colorectal+cancer+initiation.">ROS production and NF-&#954;B activation triggered by RAC1 facilitate WNT-driven intestinal stem cell proliferation and colorectal cancer initiation.</a> Cell Stem Cell. 12 (6), 761-773 (2013).</li><li>Juhasz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase+1+supports+proliferation+of+colon+cancer+cells+by+modulating+reactive+oxygen+species-dependent+signal+transduction.">NADPH oxidase 1 supports proliferation of colon cancer cells by modulating reactive oxygen species-dependent signal transduction.</a> Journal of Biological Chemistry. 292 (19), 7866-7887 (2017).</li><li>Aviello, G., Knaus, U. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+in+gastrointestinal+inflammation:+Rescue+Or+Sabotage.">ROS in gastrointestinal inflammation: Rescue Or Sabotage.</a> British Journal of Pharmacology. 174 (12), 1704-1718 (2017).</li><li>Gomes, A., Fernandes, E., Lima, J. L. F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probes+used+for+detection+of+reactive+oxygen+species.">Fluorescence probes used for detection of reactive oxygen species.</a> Journal of Biochemical and Biophysical Methods. 65 (2-3), 45-80 (2005).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Levy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+receptor+NOD2+mediates+LGR5++intestinal+stem+cell+protection+against+ROS+cytotoxicity+via+mitophagy+stimulation.">Innate immune receptor NOD2 mediates LGR5+ intestinal stem cell protection against ROS cytotoxicity via mitophagy stimulation.</a> Proceedings of the National Academy of Sciences. 117 (4), 1994-2003 (2020).</li><li>Choi, H., Yang, Z., Weisshaar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell,+real-time+detection+of+oxidative+stress+induced+in+escherichia+coli+by+the+antimicrobial+peptide+CM15.">Single-cell, real-time detection of oxidative stress induced in escherichia coli by the antimicrobial peptide CM15.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (3), 303-310 (2015).</li><li>Amri, F., Ghouili, I., Amri, M., Carrier, A., Masmoudi-Kouki, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroglobin+protects+astroglial+cells+from+hydrogen+peroxide-induced+oxidative+stress+and+apoptotic+cell+death.">Neuroglobin protects astroglial cells from hydrogen peroxide-induced oxidative stress and apoptotic cell death.</a> Journal of Neurochemistry. 140 (1), 151-169 (2017).</li><li>Ahn, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Photon+Fluorescence+Microscopy+Imaging+of+Cellular+Oxidative+Stress+Using+Profluorescent+Nitroxides.">Two-Photon Fluorescence Microscopy Imaging of Cellular Oxidative Stress Using Profluorescent Nitroxides.</a> Journal of the American Chemical Society. 134 (10), 4721-4730 (2012).</li><li>Bidaux, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+TRPM8+channel+isoform+controls+the+balance+between+keratinocyte+proliferation+and+differentiation+in+a+cold-dependent+manner.">Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a cold-dependent manner.</a> Proceedings of the National Academy of Sciences. 112 (26), 3345-3354 (2015).</li><li>Van de Bittner, G. C., Dubikovskaya, E. A., Bertozzi, C. R., Chang, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+hydrogen+peroxide+production+in+a+murine+tumor+model+with+a+chemoselective+bioluminescent+reporter.">In vivo imaging of hydrogen peroxide production in a murine tumor model with a chemoselective bioluminescent reporter.</a> Proceedings of the National Academy of Sciences. 107 (50), 21316 (2010).</li><li>Rabbani, P. S., Abdou, S. A., Sultan, D. L., Kwong, J., Duckworth, A., Ceradini, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+reactive+oxygen+species+in+a+murine+wound+model.">In vivo imaging of reactive oxygen species in a murine wound model.</a> Journal of Visualized Experiments. (141), e58450 (2018).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li></ol>

This work provides a step-by-step protocol to isolate murine jejunal crypts, culture them into 3D organoids, and analyze ROS in organoids by combining a ROS-sensitive fluorogenic probe with qualitative microscopy imaging of whole organoids and quantitative ROS measurement using flow cytometry on single cells following organoid dissociation.
The first critical step in this method is the crypts extraction procedure. Indeed, the quality of crypts preparation is the key to successful organoids for...

Reactive oxygen species (ROS) are natural by-products of cellular metabolism. They can also be actively produced by specialized enzymatic complexes such as the membrane-bound NADPH-Oxidases (NOX) and Dual Oxidases (DUOX), which generate superoxide anion and hydrogen peroxide1. By expressing antioxidant enzymes and ROS scavengers, cells can finely tune their redox balance, thereby protecting tissue homeostasis2. Although ROS can be highly toxic to the cells and damage DNA, proteins, and lipids, they are crucial signaling molecules2. In the intestinal epithelium, moderate ROS levels are required for stem and progenitor cell proliferation3; high ROS levels lead to their apoptosis4. Chronic oxidative stress is linked to many gastrointestinal diseases, such as inflammatory bowel diseases or cancer. As an example, in a mouse model of Wnt-driven intestinal cancer, elevated ROS production through activation of NADPH-oxidases was found to be required for cancer cells hyperproliferation5, 6. Defining how intestinal cells, in particular stem cells, stem cells manage oxidative stress and how the cellular environment can impact this capacity is essential to understand the etiology of this disease better7.
In a tissue, different cell types present a basal oxidative state that may vary according to their function and metabolism and the expression of varying levels of oxidant and antioxidant molecules4,7. Monitoring ROS in vivo is very challenging. Cell permeable dyes that emit fluorescence according to their redox state have been developed to visualize and measure cellular ROS in living cells and animals. However, their efficacy depends on their diffusion inside living tissues and their rapid readout, making them difficult to use in animal models8.
In the past, the study of the effect of compounds on ROS generation was done using cell lines, but this may not reflect the in vivo situation. The intestinal organoid model, developed by the group of Clevers9, enables the growth of intestinal primary cells ex vivo. Culture of intestinal crypts in matrices, in the presence of defined growth factors, leads to three-dimensional structures, called organoids (mini-gut), which reproduce the crypt-villus organization, with cells from the different epithelial lineages lining an internal lumen, and the intestinal stem cells residing in small crypts-like protrusions.
Here, taking advantage of this model, a simple method is described to study oxidative stress in primary intestinal cells at the single-cell resolution by adding a commercially available ROS-sensitive dye into the organoid culture medium.
Plate readers are often used to detect ROS production in a total population. This protocol uses flow cytometry or imaging assay to detect ROS in a particular cell type with genetically modified cells or specific antibody staining. This work involves mouse intestinal organoid culture and ROS visualization by confocal imaging and quantification by flow cytometry. Using Lgr5-GFP mice-derived small intestinal organoids, it is possible to specifically analyze the level of oxidative stress in intestinal stem cells upon different treatments. This protocol can be adapted to test the influence of exogenous molecules, such as microbiota-derived muramyl-dipeptide (MDP)10, on the ROS balance, after stimulating organoids with the selected compounds.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lgr5-EGFP-IRES-creERT2 (Lgr5-GFP)</td>
			<td>The Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Growth culture medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM F12 (DMEM/F12)</td>
			<td>ThermoFisher</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, minus vitamin A</td>
			<td>ThermoFisher</td>
			<td>12587010</td>
			<td>Stock Concentration: 50x</td>
		</tr>
		<tr>
			<td>GlutaMAX (glutamine)</td>
			<td>ThermoFisher</td>
			<td>35050038</td>
			<td>Stock Concentration: 100x</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>ThermoFisher</td>
			<td>15630056</td>
			<td>Stock Concentration: 1 M</td>
		</tr>
		<tr>
			<td>Murine EGF</td>
			<td>R&#38;D</td>
			<td>2028-EG-200</td>
			<td>Stock Concentration: 500 &#181;g/mL in PBS</td>
		</tr>
		<tr>
			<td>murine Noggin</td>
			<td>R&#38;D</td>
			<td>1967-NG/CF</td>
			<td>Stock Concentration: 100 &#181;g/mL in PBS</td>
		</tr>
		<tr>
			<td>Murine R-spondin1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td>Stock Concentration: 50 &#181;g/mL in PBS</td>
		</tr>
		<tr>
			<td>N-2 Supplement</td>
			<td>ThermoFisher</td>
			<td>17502048</td>
			<td>Stock Concentration: 100x</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S)</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td>Stock Concentration: 100x (10,000 units/mL of penicillin and 10,000 &#181;g/mL of streptomycin)</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well round bottom</td>
			<td>Corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>ball tip scissor</td>
			<td>Fine Science Tools GMBH</td>
			<td>14086-09</td>
			<td></td>
		</tr>
		<tr>
			<td>CellROX&#174; Deep Red Reagent</td>
			<td>ThermoFisher</td>
			<td>C10422</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#8217;,6-diamidino-2-ph&#233;nylindole, dichlorhydrate) (fluorgenic probe)</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td>stock at 10 mg/mL</td>
		</tr>
		<tr>
			<td>DPBS 1x no calcium no magnesium (DPBS)</td>
			<td>ThermoFisher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>FLuoroBrite DMEM (DMEM no phenol red)</td>
			<td>ThermoFisher</td>
			<td>A1896701</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td>stock at 10 mg/mL</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced, Phenol Red Free (Basement Membrane Matrix)</td>
			<td>Corning</td>
			<td>356231</td>
			<td>once received thaw o/n in the fridge, keep for 1h on ice and, make 500 mL aliquots and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 Well chambers</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine (NAC)</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Butyl hydroperoxide (tBCHP)solution (70%wt. In H2O2)</td>
			<td>Sigma</td>
			<td>458139</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1X), no phenol red (trypsin)</td>
			<td>ThermoFisher</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA, pH8.0</td>
			<td>ThermoFisher</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Rock-inhibitor to be used to minimize cell death upon tissue dissociation</td>
		</tr>
		<tr>
			<td>Programs and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Attune NxT (Flow Cytometer)</td>
			<td>ThermoFischer</td>
			<td></td>
			<td>Flow cytometer analyzer</td>
		</tr>
		<tr>
			<td>Fiji/ImageJ</td>
			<td>https://imagej.net/software/fiji/downloads</td>
			<td></td>
			<td>images generation</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>BD Bioscience</td>
			<td></td>
			<td>FACS analysis</td>
		</tr>
		<tr>
			<td>Observer.Z1</td>
			<td>Zeiss</td>
			<td></td>
			<td>confocal system</td>
		</tr>
		<tr>
			<td>Opterra (swept-field confocal)</td>
			<td>Bruker</td>
			<td></td>
			<td>confocal system</td>
		</tr>
		<tr>
			<td>high speed EMCCD Camera Evolve Delta 512</td>
			<td>Photometrics</td>
			<td></td>
			<td>confocal system</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>statistical analysis</td>
		</tr>
	</tbody>
</table>

analyzing oxidative stress in murine intestinal organoids using reactive oxygen species-sensitive fluorogenic probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

As a proof of concept of the described protocol, the crypts obtained from the Lgr5-eGFP-IRES-CreERT2 mouse line were used in which intestinal stem cells display mosaic GFP expression, which was established by Barker et al., to characterize intestinal stem cells10 initially and allow to map these cells based on their GFP expression. A model is thereby provided to compare ROS levels in a specific cell type population upon different treatments. A ROS inhibitor (NAC) was used, and an inducer (tBHP), k...

Watch this Scientific Journal Video about Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe at JoVE.com

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

Research

JoVE Journal

Biology

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...

3D Culture of Organoids from Murine Intestinal Crypts and Single Stem Cell

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research (Video) | JoVE 

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids (Video) | JoVE 

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...