Name: quantification of proliferative and dead cells in enteroids
Uploaded: 2020-01-31T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Proliferative and Dead Cells in Enteroids at JoVE.com

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 ...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;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&#38;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

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 ...

Watch this Scientific Journal Video about Quantification of Proliferative and Dead Cells in Enteroids at JoVE.com

Quantification of Proliferative and Dead Cells in Enteroids

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. ...

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