Name: Quantification of Proliferative and Dead Cells in Enteroids (Video) | JoVE Journal
Uploaded: 2020-01-31T14:00:00.000+00:00
Duration: 10 min 47 s
Description: Learn to quantify proliferative and dead cells in mouse enteroids using flow cytometry. This method enables researchers to study drug effects and cytokine responses in intestinal epithelial cells.

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5 minutes

This work is supported by The National Natural Science Foundation of China (31971062, 31900326, and 31601022), The Natural Science Foundation of Jiangsu Province (BK20190043, BK20180838), Research Fund of State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University (KF-GN-202004). The Natural Science Foundation of the Jiangsu Higher Education Institutions of China (19KJB320003), The Livelihood and Technology Program of Suzhou City (SYS2019030), and The Research Innovation Program for College Graduates of Jiangsu Province (KYCX19-1981). This work is also supported by Tang Scholar of Soochow University.

This protocol was approved by the Animal Care and Use Committee of Cambridge-Suda Genomic Resource Center (CAM-SU) at Soochow University.
1. Intestinal Organoid Isolation and Culture
<ol>
	<li>Isolation of intestinal crypts and enteroid culture
	<ol>
		<li>Euthanize an 8-week-old wild-type mouse with CO2 inhalation. Use tissue forceps and fine iris scissors to dissect out approximately 8 cm of ileum.</li>
		<li>Flush out using a syringe with gavage feeding needle with about 40 mL of ice-cold Dulbecco&#39;s phosphate-buffered saline (DPBS), then cut lengthwise with scissors and open the ileum.</li>
		<li>Cut the ileum into small (0.5&#8722;1.0 cm) pieces. Place the pieces in 5 mL of sterile ice-cold DPBS in a 15 mL conical tube, then rock for 5 min on ice.</li>
		<li>Use a pipette controller to aspirate the DPBS and replace it with 10 mL of cold buffer 1 (2 mM EDTA in DPBS). Rock for 30 min on ice.</li>
		<li>Use the pipette controller to aspirate buffer 1 and replace with 10 mL of cold buffer 2 (54.9 mM D-sorbitol, 43.4 mM sucrose in DPBS). Shake for 2&#8722;3 min by hand (~80 shakes/min).</li>
		<li>Take a 20 &#181;L droplet of buffer 2 contents after shaking and inspect under a microscope.</li>
		<li>Filter buffer 2 contents with a 70 &#181;m sterile cell strainer, then collect the filtered buffer with a 50 mL conical tube.</li>
		<li>Pipette 20 &#181;L of filtrate onto the slide to count the crypts. Transfer a sufficient volume of buffer 2 from step 1.1.7 to ensure that there are ~500 crypts/well.</li>
		<li>Spin down at 150 x g for 10 min at 4 &#176;C. Once spinning is finished, carefully aspirate the supernatant.</li>
		<li>Suspend crypts in 50 &#181;L of basement membrane matrix (e.g., Matrigel) per 500 crypts, pipette up and down (being careful to avoid bubbles), then place a 50 &#181;L droplet of basement membrane matrix/crypt mix in the center of one well in a 24 well plate. Incubate for 30 min at 37 &#176;C to polymerize basement membrane matrix.</li>
		<li>After 30 min of polymerization, carefully add 600 &#181;L of minigut media (advanced Dulbecco&#39;s modified Eagle medium [DMEM]/F12, 2 mM L-alanine-L-glutamine, pen/strep [100 units/mL], 10 mM Hepes, N2 supplement [1:100], B27 supplement [1:50] with epidermal growth factor [EGF; 50 ng/mL], Noggin [100 ng/mL], R-spondin [500 ng/mL], and Y27632 [10 &#181;M]) to each well, then return the plate to the 37 &#176;C incubator. Observe under a microscope each day, and change the media every 2&#8722;3 days.</li>
	</ol>
	</li>
	<li>Enteroid passaging 
	NOTE: For long cultures, enteroids should be split approximately each week.
	<ol>
		<li>To passage the enteroids, place the tissue culture plate on ice. Aspirate the media and add 1 mL of cold DPBS to each well. Use a P1000 tip to pipette up and down until no solid basement membrane matrix chunks remain.</li>
		<li>Pass up and down once through a 1 mL insulin syringe (27 G) and into a 15 mL conical tube. Spin down for 5 min at 150 x g at 4 &#176;C.</li>
		<li>Use pipette to remove DPBS. Resuspend in 50 &#181;L of basement membrane matrix/well.</li>
		<li>Place the plate in a 37 &#176;C incubator for 30 min to allow basement membrane matrix to polymerize. Overlay each well with 600 &#181;L of ENR media (minigut media, 50 ng/mL EGF, 100 ng/mL Noggin, 500 ng/mL R-spondin), then return the plate to the incubator.</li>
	</ol>
	</li>
</ol>
2. Flow Cytometry Analysis of EdU-positive Cells in Enteroids
NOTE: Figure 1 shows the workflow for flow cytometry analysis of EdU-positive cells in enteroids.
<ol>
	<li>Incubation of enteroids with EdU
	<ol>
		<li>Grow enteroids from step 1.2.4 for 5&#8211;7 days. Passage enteroids into a new 24 well plate in a splitting ratio of 1:2. Add 600 &#181;L of ENR medium to each well. Incubate enteroids for 4&#8722;5 days in a 37 &#176;C incubator.</li>
		<li>Set the control group (untreated enteroids) and experimental group (enteroids treated with 5 ng/mL interleukin 22 [IL-22]). Prepare at least three replicates for each group.</li>
		<li>Add EdU to the ENR medium to prepare EdU medium with a concentration of 50 &#956;M. Add 600 &#956;L of EdU medium to each well and incubate for 2 h in a 37 &#176;C incubator. Set one well of enteroids without EdU treatment as a negative control for background subtraction.</li>
	</ol>
	</li>
	<li>Harvesting of enteroids from basement membrane matrix
	<ol>
		<li>Use a pipette controller to aspirate EdU medium and wash 1x with DPBS. Add 1 mL of DPBS.</li>
		<li>Use P1000 pipette tip and pipette up and down until no solid basement membrane matrix chunks remain. Transfer to a 15 mL tube and spin down at 300 x g for 5 min. Discard the supernatant.</li>
		<li>Add 500 &#956;L of cell-dissociation enzymes (Table of Materials) and incubate for 15 min at 37 &#176;C. Use P200 pipette tip and pipette up and down to break enteroids into single cells.</li>
		<li>Add 3 mL of DMEM medium containing 10% fetal bovine serum (FBS) and repeatedly pipette with a P1000 pipette tip.</li>
		<li>Spin down at 300 x g for 5 min and aspirate the supernatant medium. Resuspend the cells with 1 mL of DPBS.</li>
	</ol>
	</li>
	<li>Fixation and permeabilization of cells
	<ol>
		<li>Transfer the cell suspension to a 1.5 mL tube, spin down at 300 x g for 5 min, and discard the supernatant.</li>
		<li>Resuspend cells with 1 mL of 4% paraformaldehyde (PFA), fix for 15 min at room temperature (RT), and spin down at 300 x g for 5 min. Aspirate the supernatant with a pipette tip, wash 1x with DPBS, and spin down to remove the supernatant.</li>
		<li>Resuspend cells with 1 mL of 0.5% nonionic surfactant. Incubate for 10 min at RT.</li>
		<li>Spin down at 300 x g for 5 min and aspirate the supernatant. Wash 1x with DPBS and spin down to remove the supernatant.</li>
	</ol>
	</li>
	<li>Detection of EdU
	<ol>
		<li>Prepare stock solutions for each component in advance: 1) working solution of the Alexa Fluor azide, 2) working solution of 1x EdU reaction buffer, and 3) 10x stock solution of EdU buffer additive.</li>
		<li>Prepare 1x EdU buffer additive by diluting the 10x solution 1:10 in deionized water. Prepare this solution fresh.</li>
		<li>Prepare reaction cocktail according to Table 1. 
		NOTE: Add the ingredients in the order listed in the table. Use the reaction cocktail within 15 min of preparation.</li>
		<li>Add 100 &#956;L of reaction cocktails to each 1.5 mL tube. Resuspend the cells, protect from light, incubate for 30 min at RT.</li>
		<li>Centrifuge at 300 x g for 5 min and aspirate the reaction solution with pipette tip gently.</li>
		<li>Add 0.5% nonionic surfactant penetrant to each tube to wash 1x at RT. Centrifuge at 300 x g for 5 min, aspirate the supernatant with pipette tip gently, and resuspend the cells in 1 mL of DPBS.</li>
	</ol>
	</li>
	<li>Analysis of cells by flow cytometry
	<ol>
		<li>Filter the resuspended cells with a 40 &#956;m strainer, then collect the filtered cells with a 15 mL conical tube. Perform FACS on-machine detection as soon as possible. 
		NOTE: If conditions are limited, the cell stained samples should be stored in the dark at 4 &#176;C and flow-tested within 3 days.</li>
		<li>Select the appropriate channel (according to the type of Alexa Fluor azide in the EdU kit; here, red channel) and voltage (here, ~150&#8211;350 V) for flow cytometry analysis.</li>
		<li>Gating strategy
		<ol>
			<li>Draw an FSC-A (x-axis) vs. SSC-A (y-axis) pseudocolor plot and distribute most of the cells to the visible range of the dot map by adjusting the voltage. Select the cell population (R1) and exclude the cell debris in the bottom left corner.</li>
			<li>From the R1 cell population, establish an FSC-A (x-axis) vs. FSC-H (y-axis) pseudocolor plot, and set the gate to select single cells (R2) to exclude cell clumps.</li>
			<li>From the R2 cell population, establish the fluorescence intensity (x-axis) vs. cell number (y-axis) plot, and use the negative control to set the gate. The region of the fluorescent signal is a positive cell region (R3). Compare the ratio of EdU-positive cells (R3) between the experimental and control groups.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Flow Cytometry Analysis of PI-positive Cells in Enteroids
NOTE: Figure 2 shows the workflow for flow cytometry analysis of PI-positive cells in enteroids.
<ol>
	<li>Incubation of enteroid cells with PI
	<ol>
		<li>Grow enteroids from step 1.2.4 for 5&#8211;7 days. Passage enteroids into a new 24 well plate in a splitting ratio of 1:2. Add 600 &#181;L of ENR medium to each well. Incubate enteroids for 4&#8722;5 days in a 37 &#176;C incubator.</li>
		<li>Set the control group (untreated) and experimental group (IL-22 treated), using at least three replicates for each group.</li>
		<li>Add PI to the ENR medium to prepare PI medium with a concentration of 3 &#956;M.</li>
		<li>Add 600 &#956;L of PI medium to each well and incubate for 30 min in a 37 &#176;C incubator. Set one well of enteroids without PI treatment as a negative control for background subtraction.</li>
	</ol>
	</li>
	<li>Harvest enteroids from basement membrane matrix following steps 2.2.1&#8722;2.2.5.</li>
	<li>Analysis of cells by flow cytometry
	<ol>
		<li>Filter the resuspended cells with a 40 &#956;m strainer and collect the filtered cells with a 15 mL conical tube.</li>
		<li>Perform FACS on-machine detection as soon as possible. 
		NOTE: If conditions are limited, the cell stained samples should be stored in the dark at 4 &#176;C and flow-tested within 3 days.</li>
		<li>Select the appropriate channel (here, red channel) and voltage (here, ~150&#8211;350 V) for flow cytometry analysis.</li>
		<li>Use the same gating strategy as described in section 2.5.3.</li>
	</ol>
	</li>
</ol>

<ol><li>Odenwald, M. A., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+intestinal+epithelial+barrier%3A+a+therapeutic+target%5BTitle%5D)+AND+%22Nature+reviews.+Gastroenterology+%26+Hepatology%22%5BJournal%5D)">The intestinal epithelial barrier: a therapeutic target.</a> Nature reviews. Gastroenterology & Hepatology. 14 (1), 9-21 (2017).</li>
<li>Buckley, A., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+Biology+of+Tight+Junction+Barrier+Regulation+and+Mucosal+Disease%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Perspectives+in+Biology%22%5BJournal%5D)">Cell Biology of Tight Junction Barrier Regulation and Mucosal Disease.</a> Cold Spring Harbor Perspectives in Biology. 10 (1), (2018).</li>
<li>Gehart, H., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tales+from+the+crypt%3A+new+insights+into+intestinal+stem+cells%5BTitle%5D)+AND+%22Nature+reviews.+Gastroenterology+%26+Hepatology%22%5BJournal%5D)">Tales from the crypt: new insights into intestinal stem cells.</a> Nature reviews. Gastroenterology & Hepatology. 16 (1), 19-34 (2019).</li>
<li>Vancamelbeke, M., Vermeire, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+intestinal+barrier%3A+a+fundamental+role+in+health+and+disease%5BTitle%5D)+AND+%22Expert+Review+of+Gastroenterology+%26+Hepatology%22%5BJournal%5D)">The intestinal barrier: a fundamental role in health and disease.</a> Expert Review of Gastroenterology & Hepatology. 11 (9), 821-834 (2017).</li>
<li>Garcia-Carbonell, R., Yao, S. J., Das, S., Guma, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dysregulation+of+Intestinal+Epithelial+Cell+RIPK+Pathways+Promotes+Chronic+Inflammation+in+the+IBD+Gut%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Dysregulation of Intestinal Epithelial Cell RIPK Pathways Promotes Chronic Inflammation in the IBD Gut.</a> Frontiers in Immunology. 10, 1094(2019).</li>
<li>Li, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vitro+and+In+Vivo+Approaches+to+Determine+Intestinal+Epithelial+Cell+Permeability%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability.</a> Journal of Visualized Experiments. (140), e57032(2018).</li>
<li>Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestinal+mucosal+barrier+function+in+health+and+disease%5BTitle%5D)+AND+%22Nature+Reviews+Immunology%22%5BJournal%5D)">Intestinal mucosal barrier function in health and disease.</a> Nature Reviews Immunology. 9 (11), 799-809 (2009).</li>
<li>Graham, W. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+MLCK1+diversion+reverses+barrier+loss+to+restore+mucosal+homeostasis%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis.</a> Nature Medicine. 25 (4), 690-700 (2019).</li>
<li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li>
<li>Sato, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growing+self-organizing+mini-guts+from+a+single+intestinal+stem+cell%3A+mechanism+and+applications%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications.</a> Science. 340 (6137), 1190-1194 (2013).</li>
<li>Friedrich, M., Pohin, M., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytokine+Networks+in+the+Pathophysiology+of+Inflammatory+Bowel+Disease%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Cytokine Networks in the Pathophysiology of Inflammatory Bowel Disease.</a> Immunity. 50 (4), 992-1006 (2019).</li>
<li>Zha, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin+22+Expands+Transit-Amplifying+Cells+While+Depleting+Lgr5+Stem+Cells+via+Inhibition+of+Wnt+and+Notch+Signaling%5BTitle%5D)+AND+%22Cellular+and+Molecular+Gastroenterology+and+Hepatology%22%5BJournal%5D)">Interleukin 22 Expands Transit-Amplifying Cells While Depleting Lgr5 Stem Cells via Inhibition of Wnt and Notch Signaling.</a> Cellular and Molecular Gastroenterology and Hepatology. 7 (2), 255-274 (2019).</li>
</ol>

The authors have nothing to disclose.

This protocol details the steps necessary for the culture of enteroids in vitro and quantification of EdU- and PI-positive cells in the enteroids by flow cytometry. There are several advantages of this strategy. First, EdU labelling is used to detect proliferating cells in enteroids. Compared with traditional BrdU assay, EdU labelling method is faster, more sensitive, and more accurate. EdU is very similar to thymine (T), which replaces thymine in DNA synthesis during cell division. Compared to the BrdU antibody, EdU is ...

A fundamental function of intestinal epithelial cells is to protect the entry of luminal contents such as pathogenic bacteria and toxins1,2. To perform such a function, intestinal stem cells continuously proliferate and differentiate into a variety of epithelial cells, including enterocytes and secretory cells, which form a barrier by forming tight connections3. The rapid renewal of intestinal epithelial cells requires strict coordination of cell proliferation, cell differentiation, and cell death4,5. Reduced cell proliferation or excessive cell death leads to epithelial damage and compromised barrier function1,6. Dysfunction of intestinal barrier has been associated with inflammatory bowel diseases7,8.
A method to culture intestinal crypts has been previously developed. Using this technique, isolated mouse crypts grow into intestinal organoids (enteroids), which have crypt-villus like structures and contain all intestinal epithelial cell lineage9,10. 5-Ethynyl-2'-deoxyuridine (EdU) is a thymidine analog that is capable of replacing thymine (T) in DNA that is undergoing replication during cell proliferation. The proliferative cells can be quickly and accurately labeled by EdU staining. Propidium iodide (PI) is an analog of ethidium bromide that releases red fluorescence upon insertion into double-stranded DNA. PI specifically detects dead cells, since it only passes through the damaged cell membrane.
In this protocol, we first describe how to isolate crypts from the murine small intestine then culture them as enteroids in vitro. We then describe how to analyze the proliferative and dead cells in enteroids by EdU and PI incorporation and flow cytometry.

<table><tbody><tr><td>15 ml centrifuge tube</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>22 G gavage needle</td><td>VWR</td><td>20068-608</td><td></td></tr><tr><td>24-well plate</td><td>Nunc</td><td>142475</td><td></td></tr><tr><td>40 mm sterile cell strainer</td><td>BD</td><td>352340</td><td></td></tr><tr><td>50 ml centrifuge tube</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>70 mm sterile cell strainer</td><td>BD</td><td>352350</td><td></td></tr><tr><td>Advanced DMEM/F-12</td><td>GIBCO</td><td>12634010</td><td></td></tr><tr><td>Attune NxT Acoustic Focusing Cytometer</td><td>Invitrogen</td><td>A24863</td><td></td></tr><tr><td>B-27 Supplement</td><td>GIBCO</td><td>17504044</td><td></td></tr><tr><td>Buffer 1</td><td></td><td></td><td>2 mM EDTA in DPBS</td></tr><tr><td>Buffer 2</td><td></td><td></td><td>54.9 mM D-sorbitol, 43.4 mM sucrose in DPBS</td></tr><tr><td>C57/B6 mice</td><td>Nanjing Biomedical Research Institute of Nanjing University</td><td></td><td></td></tr><tr><td>Cell-dissociation enzymes (TrypLE)</td><td>Life technologies</td><td>12605-010</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424R</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Click-iT Plus EdU Alexa Fluor 594 Imaging Kit</td><td>Life technologies</td><td>C10639</td><td></td></tr><tr><td>CO2 incubator</td><td>Panasonic</td><td>MCO-18AC</td><td></td></tr><tr><td>DPBS</td><td>GIBCO</td><td>14190144</td><td></td></tr><tr><td>D-sorbitol</td><td>BBI</td><td>SB0491</td><td></td></tr><tr><td>EDTA</td><td>BBI</td><td>EB0185</td><td></td></tr><tr><td>ENR media</td><td></td><td></td><td>Minigut media, 50 ng/ml EGF, 100 ng/ml Noggin, 500 ng/ml R-spondin</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>Fine Iris Scissors</td><td>Tansoole</td><td>2037454</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>GIBCO</td><td>35050-061</td><td></td></tr><tr><td>Goat Serum</td><td>Life technologies</td><td>16210-064</td><td></td></tr><tr><td>HDMEM</td><td>Hyclone</td><td>SH30243.01B</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>Minigut media</td><td></td><td></td><td>Advanced DMEM/F12, 2 mM Glutamax, Penn/Strep (100 units/ml), 10 mM Hepes, N2 supplement (1:100), B27 supplement (1:50)</td></tr><tr><td>N2 supplement</td><td>R&amp;amp;D</td><td>AR009</td><td></td></tr><tr><td>Nonionic surfactant (Triton X)</td><td>BBI</td><td>TB0198-500ML</td><td></td></tr><tr><td>Operating Scissor (12.5 cm)</td><td>Tansoole</td><td>2025785</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>sigma</td><td>158127-500g</td><td></td></tr><tr><td>Penn/Strep</td><td>Invitrogen</td><td>15140-148</td><td></td></tr><tr><td>Phase contrast microscope</td><td>Nikon</td><td>TS1000</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigma</td><td>P4170-25MG</td><td></td></tr><tr><td>Recombinant EGF</td><td>PeproTech</td><td>315-09</td><td></td></tr><tr><td>Recombinant Mouse Noggin</td><td>PeproTech</td><td>250-38</td><td></td></tr><tr><td>Recombinant Mouse R-Spondin 1</td><td>R&amp;amp;D</td><td>3474-RS-050</td><td></td></tr><tr><td>Recombinant Murine IL-22</td><td>PeproTech</td><td>210-22-10</td><td></td></tr><tr><td>Sucrose</td><td>BBI</td><td>SB0498</td><td></td></tr><tr><td>Tissue Forceps</td><td>Tansoole</td><td>2026704</td><td></td></tr><tr><td>Y-27632 2HC1</td><td>Selleck</td><td>S1049</td><td></td></tr></tbody></table>

quantification of proliferative and dead cells in enteroids

Quantification of Proliferative and Dead Cells in Enteroids

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or &ldquo;enteroids&rdquo;. Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2&rsquo;-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

Small intestinal crypts were isolated and cultured as enteroids in basement membrane matrix. Enteroids started to form buds 2 days after isolation. On day 6, enteroids had many buds with lots of debris (dead cells) in the lumen. Enteroids were ready to be passaged at this stage (Figure 3).
Numerous studies have shown that inflammatory cytokines are essential for the maintenance of intestinal epithelial homeostasis. Abnormal expression of inflammatory cytokines is ...

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. ...

quantification-of-proliferative-and-dead-cells-in-enteroids

Research

JoVE Journal

Biology

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Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

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This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

This study outlines a protocol for generating 2D monolayers of porcine organoids derived from the small and large intestines. The growth of these monolayers is marked by increasing TEER values, indicating robust epithelial integrity. Additionally, these monolayers exhibit physiological secretory responses in Ussing chamber experiments following the application of forskolin.

Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut

Here, we describe a protocol for generating apical-out intestinal organoids from standard matrix-embedded organoid cultures. It also outlines the subsequent incorporation of EdU into actively proliferating cells and the semiautomatic quantification of EdU-positive cells.

Generation of Apical-Out Intestinal Organoids and Assessment of Organoid Proliferation Rate Using EdU-Labeling

Learn to quantify proliferative and dead cells in mouse enteroids using flow cytometry. This method enables researchers to study drug effects and cytokine responses in intestinal epithelial cells.

Quantification of Proliferative and Dead Cells in Enteroids

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