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>
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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. 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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>

The authors have nothing to disclose.

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 ...

This method is a valuable tool in the field of cellular biology for assessing cellular ROS levels in living primary cells cultivated in vitro as organoids using a fluorogenic probe. The main advantage of this technique is that ROS can be qualitatively visualized in intact intestinal organoids by microscopy and quantitatively analyzed in dissociated cells by flow cytometry using 96-well plates. This method can be applied to other organoid models and alternative fluorescent sensors might be used to analyze different cellular pathways.Before applying this protocol for the first time, the experimenter should become familiar with the organoid culture and passaging. Although our protocol is well-adapted to screening approaches, beginners should start with a few samples. Demonstrating the procedure will be Sophie Dulauroy, an engineer from the laboratory.After sacrificing an 8-to-10-week-old Lgr5-GFP mouse, collect five to eight centimeters of the jejunum encompassing region between the duodenum and ileum, and place it in cold DPBS supplemented with antibiotics on ice. Next, clean the intestinal content by flushing with five to 10 milliliters of cold DPBS containing antibiotics. Then using ball tip scissors, open the intestine longitudinally, and using forceps, transfer the tissue into a Petri dish containing cold DPBS with antibiotics.Shake the tissue inside the dish to rinse it. Next, grab the intestine by aspiration using a plastic Pasteur pipette and transfer it into a 15 milliliter tube containing 10 milliliters of cold 10 millimolar EDTA. Invert the tube three times and incubate it on ice for 10 minutes.After the incubation, transfer the tissue in a tube containing 10 milliliters of DPBS and vortex for two minutes. Immediately add 10 microliters of the fraction in a Petri dish and use a microscope to assess the quality of the fraction. After two transfers in DPBS containing tubes with two-minute vortexing between each transfer, incubate the intestine in EDTA on ice for five minutes as demonstrated previously.Following three successive transfers in tubes containing DPBS with three-minute vortexing between each transfer, combine the best fractions into a 50 milliliter tube by inverting the selected tubes three times and filtering through a 70 micron cell strainer. Then spin the crypts, discard the supernatant and disrupt the pellet mechanically before adding five milliliters of cold DMEM F12. Count the number of crypts present in a 10 microliter aliquot manually under a microscope.After counting, spin the crypts again and carefully remove the supernatant. Then mechanically disrupt the pellet and add growth culture medium to the tube to obtain a concentration of 90 crypts per microliter. Next, add two volumes of undiluted basement membrane matrix or BMM to get a final concentration of 30 crypts per microliter and carefully pipette the suspension up and down without introducing air bubbles into the mix.For flow cytometry analysis, plate 10 microliters of the crypts BMM mix as a dome at the center of each well of a pre-warmed round bottom 96-well plate. And for imaging, deposit 10 microliters of the mix as a thin layer in a pre-warmed microslide eight-well chamber. After allowing the BMM to solidify at room temperature for five minutes, place the plates in an incubator at 37 degrees Celsius and 5%carbon dioxide.After 15 minutes, add 250 microliters of growth medium into each well, taking care not to detach the BMM, and place the plate in the incubator. To visualize oxidative stress by confocal microscopy, add one microliter of n-acetylcysteine stock solution in the corresponding wells of the microslide eight-well chamber plated with organoids. After a one-hour incubation, add one microliter of tert-butyl hydroperoxide stock solution in the corresponding wells and incubate for another 30 minutes.Next, add one microliter of the 1.25 millimolar dilution of the fluorogenic probe per well, followed by one microliter of 1.25 milligrams per milliliter Hoechst solution. After another 30-minute incubation, remove the medium without disturbing the BMM and gently add 250 microliters of warm DMEM without phenol red. Image the organoids using a confocal microscope equipped with a thermic chamber and gas supply that detects the fluorogenic probe.Using the positive control, set up the laser intensity and time exposure for the ROS signal, and check that this signal is lower in the negative control. Next, using an eyepiece, screen the slide to identify the organoids expressing GFP and adjust the laser intensity. Set up a z-stack of 25 micrometers and define positions to obtain a stitched image of the whole organoid to get a section of the organoids showing one layer of cells.Add one microliter of the n-acetylcysteine stock solution in the negative control wells of the 96-well round bottom plate containing the organoids. After a one-hour incubation, add one microliter tert-butyl hydroperoxide stock solution in the corresponding wells and incubate for 30 minutes. Then using a multi-channel pipette, remove the medium without disturbing the attached BMM and transfer it to another 96-well round bottom plate.Keep this plate aside. Next, add 100 microliters of trypsin and using a multi-channel pipette, pipette up and down five times to destroy the BMM. After a short five-minute incubation, dissociate the organoids by pipetting up and down a second time.Spin the plate and discard the supernatant by inverting the plate. Add the medium previously collected in another 96-well plate back into the corresponding wells and resuspend the cells by pipetting up and down five times. Next, add one microliter of the fluorogenic probe and incubate for 30 minutes.Then spin the plate again and resuspend the cells with 250 microliters of DAPI solution. Transfer the samples in flow cytometry tubes and keep the tubes on ice. Optimize the forward and side scatter voltage settings on unstained control and laser voltages for each fluorophore using monostained samples.Then using an appropriate gating strategy, collect a minimum of 20, 000 events. Representative confocal images of ROS staining and organoids showed that in the presence of the NAC inhibitor, only the signal from the dead cells contained in the lumen of the organoid is visible. In the non-treated organoid, the basal ROS levels can be seen, particularly in GFP positive cells, proving that stem cells produce higher ROS than differentiated cells.GFP positive cells present a more significant cytoplasmic signal with the inducer in the presence of the fluorogenic probe, demonstrating that ROS levels increase particularly in stem cells after treatment. Flow cytometry analysis of ROS production in the intestinal organoids shows that basal ROS levels decrease after stimulation with inhibitor and increase after challenge with inducer. Cells pretreated with inhibitor and then stimulated with inducer present lower levels than those stimulated with inducer alone.A similar analysis on stem cells gated as GFP positive shows a 3.5 fold decrease in ROS level upon inhibitor treatment and a fourfold increase upon inducer treatment over non-stimulated cells. When attempting this procedure, users should remember to limit cell stress during organoid manipulation and dissociation. Additionally, positive and negative controls should always be included when analyzing ROS.Following this procedure, the analysis can be complemented by immunofluorescence staining on fixed intact organoids, cell sorting, and gene expression analysis to get additional insights on ROS regulation. After watching this video, you should know how to grow murine intestinal organoids, perform live imaging analysis of intact organoids, and process intestinal organs for flow cytometry analysis.

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JoVE Journal

Biology

3.6K visualizações.  Institut Pasteur. Este método é uma ferramenta valiosa no campo da biologia celular para avaliar os níveis de ROS celulares em células primárias vivas cultivadas in vitro como organoides usando uma sonda fluorogênica. A principal vantagem dessa técnica é que o ROS pode ser qualitativamente visualizado em organoides intestinais intactos por microscopia e analisado quantitativamente em células dissociadas por citometria de fluxo usando placas de 96 poços. Este método pode ser aplicado a outros modelos...