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 500 ng/mL of mouse R-Spondin1 (see Table of Materials). Leave the medium at room temperature (RT) during the crypt&#39;s extraction. 
	NOTE: Freeze the unused medium in aliquots at -20 &#176;C. Avoid freeze and thaw.</li>
	<li>Fill a 50 mL tube with 40 mL of Advanced DMEM/F-12 and keep it on ice. 
	NOTE: Keep the unused medium at 4 &#176;C. It will be used for organoids passaging.</li>
	<li>Pre-warm the cell culture plates (&#181;-Slide 8 well chambers and/or 96-well round bottom) in the incubator at 37 &#176;C.</li>
	<li>Thaw basement membrane matrix (BMM) (see Table of Materials) aliquots on ice (before starting the protocol or at least 1 h before plating crypts). 
	NOTE: The BMM will quickly solidify if not kept cold.</li>
	<li>Prepare washing/flushing solution adding 1% penicillin-streptomycin solution to DPBS (DPBS-P/S).</li>
	<li>Fill a 100 mm petri-dish with 10 mL of cold DPBS-P/S. Fill six 15 mL tubes with 10 mL of DPBS and label them from F1 to F6.</li>
	<li>Prepare 30 mL of 10 mM EDTA solution by dilution from 0.5 M EDTA in DPBS. Fill two 15 mL tubes with 10 mL of 10 mM EDTA, and label them E1 and E2.</li>
	<li>Keep all solutions pre-cooled at 4&#176;C and keep them on the ice during the procedure.</li>
</ol>
2. Intestinal organoids culture
<ol>
	<li>Sacrifice a 8-10 weeks-old Lgr5-EGFP-IRES-creERT2 (Lgr5-GFP) mouse according to the national rules and regulations.</li>
	<li>Collect 5-8 cm of the jejunum encompassing the region between the duodenum (5 cm from the stomach) and the ileum (10 cm from the cecum) and keep in cold DPBS-P/S on ice.</li>
	<li>Clean the intestinal content by flushing with 5-10 mL of cold DPBS-P/S. 
	NOTE: Home-made flushing syringes can be obtained by plugging a 200 &#181;L tip onto a 10 mL syringe nozzle.</li>
	<li>Open the intestine longitudinally using ball tip scissors (see Table of Materials) (to prevent damaging the tissue).</li>
	<li>Using forceps, transfer the tissue into a petri dish containing cold DPBS-P/S at room temperature and shake it to rinse.</li>
	<li>With a plastic Pasteur pipette, grab the intestine by aspiration and transfer it into a 15 mL tube labeled E1 containing 10 mL cold 10 mM EDTA.</li>
	<li>Invert the tube 3 times and incubate on ice for 10 min.</li>
	<li>Using a plastic Pasteur pipette, transfer the tissue in tube F1 containing 10 mL DPBS. Vortex for 2 min (on normal vortex, holding the tube by hand and ensuring that the intestine swirls nicely).</li>
	<li>Put 10 &#181;L of the fraction in a petri dish and assess the quality of the fraction under a microscope. 
	NOTE: All vortex steps are performed at maximum speed, and the quality of each fraction should be assessed under the microscope (Figure 1).</li>
	<li>With a plastic Pasteur pipette, grab the intestine by aspiration and transfer it in tube F2 containing 10 mL DPBS and vortex for 2 min.</li>
	<li>Repeat step 2.10, transferring the tissue in tube F3 containing 10 mL DPBS and vortex for 2 min.</li>
	<li>Repeat EDTA incubation as in step 2.6, transferring the tissue in tube E2 containing 10 mM EDTA.</li>
	<li>Invert the tube 3 times and incubate on ice for 5 min.</li>
	<li>Repeat step 2.10, transferring the tissue in tube F4 containing 10 mL DPBS and vortex for 3 min.</li>
	<li>Repeat step 2.14, transferring the tissue in tube F5 containing 10 mL DPBS and vortex for 3 min.</li>
	<li>Repeat step 2.15, transferring the tissue in tube F6 containing 10 mL DPBS and vortex for 3 min.</li>
	<li>Combine the best fractions filtering by gravity through a 70 &#181;m cell strainer into a 50 mL tube (on ice) to eliminate villi and significant debris. 
	NOTE: Usually, F5 and F6 are the fractions containing numerous crypts and less debris.</li>
	<li>Spin the crypts at 150 x g at 4 &#176;C for 3 min.</li>
	<li>Empty the tube, disrupt the pellet mechanically, and add 5 mL of cold DMEM/F12.</li>
	<li>Put 10 &#181;L of the suspension in a petri dish and count the number of crypts present in the aliquot manually under a microscope. 
	NOTE: Do not count single cells or small debris.</li>
	<li>Calculate the volume (V) of crypts suspension required in &#181;L, considering that 300 crypts are plated per well, W is the number of wells, and N is the number of crypts counted out of 10 &#181;L of the suspension. Transfer the crypts to a new 15 mL tube. 
	NOTE: V = 300 x W x 10/N.&#160;If a small volume is used in the planned experiment, a 1.5 mL centrifuge tube can be used.</li>
	<li>Spin the crypts at 200 x g at 4 &#176;C for 3 min.</li>
	<li>Carefully remove the supernatant using a pipette.</li>
	<li>Mechanically disrupt the pellet and gently add growth culture medium to obtain a concentration of 90 crypts/&#181;L.</li>
	<li>Add 2 volumes of undiluted BMM to have a final concentration of 30 crypts/&#181;L. Carefully pipette up and down without introducing air bubbles into the mix. 
	NOTE: Always keep the tube on ice to avoid BMM solidification.</li>
	<li>Plate 10 &#181;L of the crypts/BMM mix into each well. For Flow cytometry analysis, use round-bottom 96-well plates. Distribute 10 &#181;L at the center of each well as a dome. For imaging, use &#181;-slide 8 well (see Table of Materials) and deposit the 10 &#181;L as a thin layer. 
	NOTE: Plate the organoids as a thin layer for the imaging assay to enable their in-depth imaging.</li>
	<li>Leave the plate for 5 min at RT to allow the BMM to solidify. Place the plate in the incubator at 37 &#176;C and 5% CO2 for 15 min.</li>
	<li>Add 250 &#181;L of growth medium into each well, taking care not to detach the BMM.</li>
	<li>Place the plates in the incubator at 37 &#176;C and 5% CO2.</li>
	<li>Perform the ROS analysis between days 4 and 6 of culture. Otherwise, change the medium and split the organoids after the appearance of several and long budding structures and when dead cells accumulate into the organoids lumens.</li>
</ol>
3. Organoids passaging
<ol>
	<li>Start passaging small intestinal organoids from the 6th day of culture, when significant budding structures have formed, and the organoids lumens have become dark. 
	NOTE: The organoid&#39;s lumen becomes dark due to the accumulation of dead cells, debris, and mucus. Avoid letting the organoids overgrow before splitting. The splitting ratio depends on the organoids&#39; growth. Passaging the organoids with a ratio of 1:2 at day 6 and 1:3 at day 10 is recommended.</li>
	<li>Fill a 15 mL tube with 4 mL of cold Advanced DMEM/F-12 and keep it on ice. 
	NOTE: Here, volumes for a 96-well culture plate are provided. If a different format is used, adjust the volume accordingly.</li>
	<li>Carefully aspirate the medium with a pipette or a vacuum pump from the wells without touching the BMM domes, and discard it.</li>
	<li>Add 100 &#181;L of cold Advanced DMEM/F-12 per well. Pipette up and down to break the BMM and transfer the content of the well into the 15 mL tube.</li>
	<li>Wash the well with 200 &#181;L cold Advanced DMEM/F-12 and collect it in the same tube. 
	NOTE: If passaging multiple wells from the same experimental condition, the contents of the wells can be pooled in the same 15 mL collecting tube.</li>
	<li>Spin the 15 mL collecting tube at 100 x g for 5 min at 4&#176;C.</li>
	<li>Discard the supernatant and add 1mL of cold Advanced DMEM/F-12 to the pellet. Using a P1000 tip, take up a P10 tip (without filter) and pipette up and down at least 20 times.</li>
	<li>Add 4 mL of cold Advanced DMEM/F-12 to the tube. Spin at 300 x g for 5 min at 4&#176;C.</li>
	<li>Aspirate the supernatant with a pipette or a vacuum pump without disturbing the pellet. Then, disrupt the pellet mechanically.</li>
	<li>Add BMM diluted in growth culture medium (2:1 ratio). Carefully pipette up and down without introducing air bubbles into the mix.</li>
	<li>Plate 10 &#181;L of the crypts/BMM mix into each well.</li>
	<li>Keep the plate for 5 min at RT to allow the BMM to solidify. Place the plate in the incubator at 37 &#176;C and 5% CO2 for 15 min.</li>
	<li>Add 250 &#181;L of growth medium into each well. 
	NOTE: Be careful not to detach the BMM.</li>
	<li>Place the plates in the incubator at 37 &#176;C and 5% CO2.</li>
</ol>
4. Preparation of reagents and materials to assess oxidative stress in intestinal organoids
<ol>
	<li>Prepare a 250 mM stock solution of inhibitor N-acetylcysteine (NAC) (see Table of Materials), resuspend 10 mg with 245 &#181;L of DPBS. Use at 1 mM final concentration.</li>
	<li>Prepare a 50 mM stock solution of inducer Tert-butyl hydroperoxide (tBHP), 70% in water, dilute 3.22 &#181;L with 496.8 &#181;L of DPBS. Use at 200 &#181;M final concentration.</li>
	<li>For the Flow cytometry study, prepare a 250 &#181;M working solution of a fluorogenic probe (see Table of Materials) by diluting the stock solution 1/10 in DMSO. Use at 1 &#181;M final concentration. 
	NOTE: As indicated in the manufacturer&#39;s instructions, the fluorogenic probe is sensitive to light and oxygen. Stocks and aliquots should not be open and close too many times.</li>
	<li>For the imaging study, prepare a 1.25 mM working solution of the fluorogenic probe by diluting the stock solution 1/2 in DMSO. Use at 5 &#181;M final concentration.</li>
	<li>Prepare a final solution of 0.1 &#181;g/mL DAPI in DPBS, to be used for dead cell discrimination in the Flow cytometry assay.</li>
	<li>Dilute Hoechst 33342 to 1.25 mg/mL in DPBS. Use at 5 &#181;g/mL final concentration to be used for nuclear staining in the imaging assay.</li>
	<li>Warm DMEM without phenol red at 37 &#176;C. 
	&#8203;NOTE: These steps describe using negative and positive controls that must be included in any assays, using the conditions indicated in Figure 2A. The assay can be used to test anti- or pro-oxidant compounds. The steps are the same, and the only difference is when the compounds are added before using the fluorogenic dye.</li>
</ol>
5. Visualization of oxidative stress in 3D organoids by confocal microscopy
<ol>
	<li>Take the organoids plated in the &#181;-Slide 8 well chambers and add 1 &#181;L NAC stock solution in the corresponding wells to obtain a final concentration of 1 mM.</li>
	<li>Incubate for 1 h at 37 &#176;C and 5% CO2.</li>
	<li>Add 1 &#181;L tBHP stock solution in the corresponding wells to obtain a final concentration of 200 &#181;M.</li>
	<li>Incubate for 30 min at 37 &#176;C and 5% CO2.</li>
	<li>Add 1 &#181;L per well of the 1.25 mM dilution of the fluorogenic probe to obtain a final concentration of 5 &#181;M.</li>
	<li>Add 1 &#181;L per well of the 1.25 mg/mL dilution of Hoescht to obtain a final concentration of 5 &#181;g/mL.</li>
	<li>Incubate for 30 min at 37 &#176;C and 5% CO2.</li>
	<li>Remove the medium without disturbing the BMM. Gently, add 250&#181;L of warm DMEM without phenol red. 
	NOTE: If a long-term acquisition is planned, add growth factors compounds to DMEM without phenol red.</li>
	<li>Image the organoids using a confocal microscope equipped with a thermic chamber and gas supply that detects the fluorogenic probe (ROS). 
	NOTE: The excitation/emission (ex/em) for the fluorogenic probe is 644/665, ex/em for Hoechst (nuclei) is 361/486, and ex/em for GFP (intestinal stem cell from the Lgr5-GFP mice) is 488/510. A 63x oil immersion objective is used to detect signals in stem cells. Do not change laser settings between samples. A 20x objective might be used to allow an overview of ROS production.</li>
	<li>Use the positive control to set up laser intensity and time exposure for the ROS signal and check that this signal is lower in the negative control.</li>
	<li>Using eyepiece screen the slide to identify the GFP organoids expressing and&#160;adjust laser intensity. 
	NOTE: This step is manually performed.</li>
	<li>Define positions to obtain a stitched image of the whole organoid. Setup a z-stack of 25 &#181;m (step size 5 &#181;m) to get a section of the organoids showing one layer of cells. 
	NOTE: Refer to the microscope user manual to optimize the setup. Using living cells, the acquisition should be done within 1 h after the end of the incubation.</li>
	<li>Open the images in an open-source image processing software (see Table of Materials).</li>
	<li>Go through the z-stack and choose the section in which the middle of the organoids is well represented and create a new image with the selected area.</li>
	<li>Quantify the images as per steps 5.15.1 - 5.15.5.
	<ol>
		<li>Select the freehand line tool.</li>
		<li>Draw a line following the nuclei. 
		NOTE: Select only regions presenting GFP-positive cells if only stem cells are analyzed.</li>
		<li>Increase the line width to cover the cell layer with the line without including the luminal debris.</li>
		<li>Select the channel for the ROS signal and measure the fluorescence intensity in the selected region and annotate the values.</li>
		<li>Draw a line where there is no signal and measure the fluorescent intensity of the background that will be subtracted to the previous value to get the final intensity.</li>
	</ol>
	</li>
</ol>
6. Quantification of the oxidative stress on the dissociated organoids using flow cytometer
<ol>
	<li>Add 1 &#181;L NAC stock solution in the wells for negative controls to obtain a final concentration of 1 mM. 
	NOTE: Use the organoids plated in the 96-well round-bottom plates.</li>
	<li>Incubate for 1 h at 37 &#176;C and 5% CO2.</li>
	<li>Add 1 &#181;L tBHP stock solution in the corresponding wells to obtain a final concentration of 200 &#181;M.</li>
	<li>Incubate for 30 min at 37 &#176;C and 5% CO2.</li>
	<li>With a multichannel pipette, remove the medium without disturbing the attached BMM and transfer it to another 96-well round bottom plate. Keep this plate aside.</li>
	<li>Add 100 &#181;L of trypsin, and with a multichannel pipette, pipette up and down at least five times to destroy the BMM.</li>
	<li>Incubate for not more than 5 min at 37 &#176;C and 5% CO2.</li>
	<li>With a multichannel pipette, pipette up and down at least five times to dissociate the organoids.</li>
	<li>Spin at 300 x g for 5 min at RT.</li>
	<li>Discard the supernatant by inverting the plate. Add back the medium collected in step 6.3 to the corresponding wells and resuspend the cells by pipetting up and down 5 times.</li>
	<li>Add the fluorogenic probe at the final concentration of 1 &#181;M. Add 1 &#181;L per well from the 250 &#181;M dilution and incubate for 30 min at 37 &#176;C and 5% CO2. 
	NOTE: Do not add the fluorogenic probe to the wells needed for the instrument&#39;s settings (Figure 2B).</li>
	<li>Spin at 300 x g for 5 min at RT.</li>
	<li>Resuspend the cells with 250 &#181;L of 0.1 &#181;g/mL DAPI solution. Transfer the samples in the proper Flow cytometry tubes, keep the tubes on ice, and proceed with the analysis. 
	NOTE: Add PBS instead of DAPI to the wells needed for the instrument&#39;s settings (Figure 2B).</li>
	<li>Optimize the forward and side scatter voltage settings on unstained control and laser voltages for each fluorophore using mono-stained samples.</li>
	<li>Using an appropriate gating strategy (Figure 4A), collect a minimum of 20,000 events. 
	NOTE: 50,000 events are preferred. Detailed acquisition settings vary according to the instrument used.</li>
</ol>

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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 ...

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Research

JoVE Journal

Biology

3.7K Views.  Institut Pasteur. 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.

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

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 eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

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 oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

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 factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

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

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

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

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well 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 inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of 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 compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

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

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 infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

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

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

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

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

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

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

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

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

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

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.