The authors would like to thank Dr. Sheryl Coutermarsh-Ott, Dylan McDaniel and Bettina Heid for technical discussions. The authors thank Dr. Nanda Nanthakumar for providing the Caco-2 cells. The authors also thank The Multicultural Academic Opportunities Program (MAOP) at Virginia Tech. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Award K01DK092355 (to I.C.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All research was approved and conducted under Virginia Tech IACUC guidelines
1. Prepare R-Spondin1 Conditioned Media From HEK293T-Rspo1 Cell Line
<ol>
	<li>Generation of HEK293T-Rspondin1 cells has been previously described10. Seed HEK293T-Rspondin1 secreting cells at 5-10% confluency, approximately 8 x 105-1.7 x 106 cells, in a T-175 flask with 40 ml of 1x Dulbecco&#39;s Modified Eagle Medium (DMEM) + 10% Fetal Bovine Serum (FBS) as the growth media, and incubate at 37 &#176;C +...

<ol>
	<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>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><li>Boj, S. F., 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=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Huch, M., 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+culture+of+genome-stable+bipotent+stem+cells+from+adult+human+liver.">Long-term culture of genome-stable bipotent stem cells from adult human liver.</a> Cell. 160 (1-2), 299-312 (2015).</li><li>Lancaster, M. 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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501 (7467), 373-379 (2013).</li><li>Freshney, R. I., Freshney, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of epithelial cells. 2nd edn. , (2002).</li><li>Vachon, P. H., 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=Differentiation+state-selective+roles+of+p38+isoforms+in+human+intestinal+epithelial+cell+anoikis.">Differentiation state-selective roles of p38 isoforms in human intestinal epithelial cell anoikis.</a> Gastroenterology. 123 (6), 1980-1991 (2002).</li><li>Hynds, R. E., Giangreco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+the+relevance+of+human+stem+cell-derived+organoid+models+for+epithelial+translational+medicine.">Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine.</a> Stem Cells. 31 (3), 417-422 (2013).</li><li>Kaiko, G. E., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-microbe+interactions+shaping+the+gastrointestinal+environment.">Host-microbe interactions shaping the gastrointestinal environment.</a> Trends Immunol. 35 (11), 538-548 (2014).</li><li>Kim, K. 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=Mitogenic+influence+of+human+R-spondin1+on+the+intestinal+epithelium.">Mitogenic influence of human R-spondin1 on the intestinal epithelium.</a> Science. 309 (5738), 1256-1259 (2005).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction:+twenty-something+years+on.">The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.</a> Nat Protoc. 1 (2), 581-585 (2006).</li><li>Allen, I. C., 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=NLRX1+protein+attenuates+inflammatory+responses+to+infection+by+interfering+with+the+RIG-I-MAVS+and+TRAF6-NF-kappaB+signaling+pathways.">NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-kappaB signaling pathways.</a> Immunity. 34 (6), 854-865 (2011).</li><li>Zhang, Y. G., Wu, S., Xia, Y., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella-infected+crypt-derived+intestinal+organoid+culture+system+for+host-bacterial+interactions.">Salmonella-infected crypt-derived intestinal organoid culture system for host-bacterial interactions.</a> Physiol Rep. 2 (9), (2014).</li><li>Grabinger, 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=Ex+vivo+culture+of+intestinal+crypt+organoids+as+a+model+system+for+assessing+cell+death+induction+in+intestinal+epithelial+cells+and+enteropathy.">Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy.</a> Cell Death Dis. 5, 1228 (2014).</li><li>Slater, T. F., Sawyer, B., Straeuli, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+Succinate-Tetrazolium+Reductase+Systems.+Iii.+Points+of+Coupling+of+Four+Different+Tetrazolium+Salts.">Studies on Succinate-Tetrazolium Reductase Systems. Iii. Points of Coupling of Four Different Tetrazolium Salts.</a> Biochim Biophys Acta. 77, 383-393 (1963).</li><li>Parry, M. F., Neu, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+N-acetylcysteine+on+antibiotic+activity+and+bacterial+growth+in+vitro.">Effect of N-acetylcysteine on antibiotic activity and bacterial growth in vitro.</a> J Clin Microbiol. 5 (1), 58-61 (1977).</li></ol>

The culture and maintenance of intestinal organoids is a procedure that can be mastered by any individual with adequate tissue culture technique. There are subtleties in passaging when compared to growing cells in a more conventional monolayer, but these subtleties are not difficult to overcome. The critical steps of this method involve being able to grow the organoids to a high enough density for optimal seeding. Experiments must be scaled down with organoids as large seeding densities that can commonly be achieved with...

The ability to harvest and culture primary organoids have been described for small intestine, colon, pancreas, liver and brain and are exciting advances germane to understanding a more physiologically representative phenomena for tissue biology1-5. The first methods describing the culture and maintenance of small intestinal organoids was reported by Sato et al. out of the lab of Hans Clevers1. Prior to this method, harvesting and culture of primary intestinal epithelial cells proved to be limited and ineffective in sustaining epithelial cell growth. Methods included dissociation of tissue via incubation with enzymes, such as collagenase and dispase, which would ultimately lead to the outgrowth of intermixed primary fibroblast cells6. These conditions would also be time restricted in sustaining the epithelial cell culture. Minimal to no epithelial cell niche would form, as the epithelial cells would enter apoptosis due to the lack of appropriate growth factors or loss of contact integrity, termed anokis7. The advent of the 3D-organoid culture system has provided a method to culture primary intestinal cells containing a spectrum of intestinal cell types in sustained culture1. These epithelial organoids have advantages over cell lines being that they are composed of several differentiated cells, and better mimic the organ they are derived from in vivo8. The process to ultimately &#34;grow a mini gut in a dish&#34; has proven to be a valuable tool for assessing the response of intestinal epithelium under different stimuli. Investigating the interaction of primary intestinal cells with microbial pathogen associated molecular patterns (PAMPs) is relevant to the field of immunology as these molecular patterns can regulate diverse responses from both host and microbe9. Not only can investigators now explore these interactions with mouse organoids, but they can be cultured from humans as well2. This technology has the potential to dramatically alter personalized medicine and it is tempting to speculate about advances that this technique will make possible in the near future.
The overall goal of this method is to provide a protocol for the culture, expansion, and treatment of intestinal organoids with a variety of stimuli. Such stimuli can ultimately range from vaccines, bacterial PAMPs, live pathogens, gastrointestinal (GI) and cancer therapeutics. The isolation and culture of mouse intestinal organoids has been adapted from Sato et al. Though there are slight deviations from the original method, the end product being organoid culture is still achieved when following this protocol. This method is focused on describing an adequate technique for proper normalization when working with non-homogenous cell structures, which must be taken into consideration when conducting an assay based on cell number.

<table>
	<tbody>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11050</td>
			<td>(Section 1,3,6) Or equivalent brand</td>
		</tr>
		<tr>
			<td>Sorvall Legend XTR Centrifuge</td>
			<td>Thermo</td>
			<td></td>
			<td>(Section 1,3)</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GE Healthcare</td>
			<td>Sh30243.01</td>
			<td>(Section 1,6) For Caco-2 and HEK293 Rspondin1 cells</td>
		</tr>
		<tr>
			<td>HEK293T-Rspondin1 secreting cell line</td>
			<td></td>
			<td></td>
			<td>(Section 1) Described and modified from Kim, K.A. et al. Lentiviral particles contained RSPO1(NM_138683) ORF cDNA cloned into a pReceiver-Lv105 backbone custom ordered and purchased from GeneCopoeia.&#160;</td>
		</tr>
		<tr>
			<td>50 ml conical tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>(Section 1) Or equivalent brand</td>
		</tr>
		<tr>
			<td>T-175 Flask</td>
			<td>Corning</td>
			<td>431079</td>
			<td>(Section 1) Or equivalent brand&#160;</td>
		</tr>
		<tr>
			<td>Protein Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td>(Section 2,3,4,5,6) Matrigel Growth Factor Reduced&#160;</td>
		</tr>
		<tr>
			<td>HyClone Dulbecco&#39;s (DPBS)</td>
			<td>GE Healthcare</td>
			<td>SH30264.01</td>
			<td>(Section 2,3)</td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td>(Section 2,3) Advanced DMEM/F12</td>
		</tr>
		<tr>
			<td>Corning 24 Well TC Plates</td>
			<td>Corning</td>
			<td>3524</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>N2 Supplement 100x&#160;</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>B27 without vitamin A 50x&#160;</td>
			<td>Life Technologies</td>
			<td>12587-010&#160;</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td>(Section 2)</td>
		</tr>
		<tr>
			<td>Glutamine Supplement (Glutamax)</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td>(Section 2) Can Combine with Advanced DMEM/ F12</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Life Technologies</td>
			<td>15630-080</td>
			<td>(Section 2) Can Combine with Advanced DMEM/ F12&#160;</td>
		</tr>
		<tr>
			<td>10ml Serological Pipet</td>
			<td>Falcon</td>
			<td>357551</td>
			<td>(Section 2) Or equivalent brand</td>
		</tr>
		<tr>
			<td>Murine Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>(Section 2) Stock = 100 mg/ml</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td>(Section 2) Stock = 1M</td>
		</tr>
		<tr>
			<td>Recombinant Mouse EGF</td>
			<td>Biolegend</td>
			<td>585608</td>
			<td>(Section 2) Stock = 500 mg/ml</td>
		</tr>
		<tr>
			<td>Rocker Variable</td>
			<td>Bioexpres</td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>dissecting scissors</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>dissecting tweezers</td>
			<td></td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>25 ml Serological Pipet</td>
			<td>Falcon</td>
			<td></td>
			<td>(Section 3)</td>
		</tr>
		<tr>
			<td>EDTA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SLBB9821</td>
			<td>(Section 3) 0.5M or alternative TC grade EDTA</td>
		</tr>
		<tr>
			<td>Sterile Petri Dish 100mm x 15mm</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td>(Section 3) Or equal sized TC dish</td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td>(Section 4)</td>
		</tr>
		<tr>
			<td>Precision Glide Needle</td>
			<td>Becton Dickinson</td>
			<td>305120</td>
			<td>(Section 4) 23G x 1 1/4 (0.6mm x 30mm)</td>
		</tr>
		<tr>
			<td>Flagellin from Bacillus subtilis</td>
			<td>Invivogen</td>
			<td>tlrl-bsfla&#160;</td>
			<td>(Section 5,6)</td>
		</tr>
		<tr>
			<td>Listeria monocytogenes</td>
			<td>ATCC</td>
			<td>19115</td>
			<td>(Section 5,6)&#160;(Murray&#160;et&#160;al.)&#160;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629-1EA</td>
			<td>(Section 5,6)Or equivalent brand</td>
		</tr>
		<tr>
			<td>BBL Brain Heart Infusion Agar</td>
			<td>Becton Dickinson</td>
			<td>211065</td>
			<td>(Section 5)</td>
		</tr>
		<tr>
			<td>Bacto Brain Heart Infusion</td>
			<td>Becton Dickinson</td>
			<td>237500</td>
			<td>(Section 5)</td>
		</tr>
		<tr>
			<td>Caco-2</td>
			<td>ATCC</td>
			<td>HTB-37</td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>gibco</td>
			<td>25200056</td>
			<td>(section 6)</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412-4</td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>SpectraMax M5</td>
			<td>Molecuar Devices</td>
			<td></td>
			<td>(Section 6)</td>
		</tr>
		<tr>
			<td>96 Well Assay Plate</td>
			<td>Corning</td>
			<td>3603</td>
			<td>(Section 6) Black Plate, Clear Bottom TC treated</td>
		</tr>
		<tr>
			<td>Nuclear Staining Dye</td>
			<td>Life Technologies</td>
			<td>H1399</td>
			<td>(section 6) Hoechst 33342</td>
		</tr>
		<tr>
			<td>T-75 Flask</td>
			<td>Corning</td>
			<td>430641</td>
			<td>(Section 6) Or equivalent brand</td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td>(Section1,3) Or equivalent brand</td>
		</tr>
		<tr>
			<td>1.7 ml polypropylene tube</td>
			<td>Bioexpress</td>
			<td>C-3262-1</td>
			<td>Or equivalent brand</td>
		</tr>
		<tr>
			<td>Quick-RNA MiniPrep</td>
			<td>Zymo Research</td>
			<td>R1054</td>
			<td>Or equivalent brand</td>
		</tr>
		<tr>
			<td>TNF-alpha&#160;</td>
			<td>Applied Biosystems</td>
			<td>Mm 00443260_g1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-6&#160;</td>
			<td>Applied Biosystems</td>
			<td>(Mm 00446190_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-1beta</td>
			<td>Applied Biosystems</td>
			<td>Mm 00434228_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>IL-18</td>
			<td>Applied Biosystems</td>
			<td>Mm 00434225_m1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>18s</td>
			<td>Applied Biosystems</td>
			<td>Hs 99999901_s1</td>
			<td>Taqman gene expression assay kit</td>
		</tr>
		<tr>
			<td>7500 Fast Real Time PCR System</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nexus gradient Mastercycler</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Fast Universal PCR Master Mix</td>
			<td>Life Technologies</td>
			<td>4352042</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity cDNA Reverse Transcription Kit</td>
			<td>Life Technologies/Applied Biosystems</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Optical 96-Well Reaction Plate, 0.1 mL</td>
			<td>Life Technologies/Applied Biosystems</td>
			<td>4346907</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-Spondin 1 Protein</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS-050</td>
			<td>500 ng/ml</td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C7559</td>
			<td></td>
		</tr>
	</tbody>
</table>

the ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

When following this protocol to cultivate intestinal organoids, characteristic sphere shaped organoids will be present after harvesting. The addition of R-spondin1 conditioned media daily will initiate the growth and budding of the organoids. The growth of organoids is shown in Figure 1A-F, and is representative of intestinal organoids on days 1, 2, 4, 5, 6 and day 14. Figure 1F represents the non-homogeneous growth chara...

Watch this Scientific Journal Video about The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids at JoVE.com

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

The overall goal of this procedure is to measure the effects of pathogenic challenge on small intestine organoids with an emphasis on normalization techniques against the total cell number.This method could help answer key questions in the innate as well as mucosal immunology fields by providing a means to investigate host pathogen interactions of bacteria with intestinal epithelial cells in vitro.Essential to this procedure is the method for normalization against the total cell number, a necessity when measuring organoid secreted proteins via techniques such as ELISA.To harvest mouse small intestinal crypts for organoid culture, begin by using a sterile glass slide to gently scrape away the villi from the luminal surface of the small intestine.Next, use dissecting scissors to cut the tissue into 1-2 centimeter length strips.And place the strips into a 50 milliliter conical tube containing 10 milliliters of ice cold PBS.Mix the contents by gentle inversion and allow the tissue contents to settle to the bottom of the tube.Then, aspirate the PBS and wash the strips three more times in 10 milliliters of fresh PBS, each time in the same manner.At the end of the final wash, place the tube on ice for ten minutes.Then, replace the PBS with 25 milliliters of PBS supplemented with 2 millimolar EDTA, in an ice bucket on a rocking platform for 45 minutes.At the end of the incubation, allow the tissue strips to settle to the bottom of the tube and replace the PBS-EDTA with 10 milliliters of PBS supplemented with 10%FBS.Shake the tube vigorously by hand ten times.Then, allow the tissue to settle to the bottom of the tube and transfer the supernatant to a 15 milliliter conical tube labeled, Fraction 1.Agitate the tissues in PBS FBS five more times.Transferring the supernatant into a new fraction tube each time.After the last agitation, centrifuge all of the samples and resuspend the pellets in five milliliters of prewarmed DMEM/F12 with no added growth factors.Now, spin down the samples again and aspirate four milliliters of the supernatant from each tube.Resuspend the pellets in the last milliliter of remaining medium and use 20 microliter aliquots from each fraction to visualize the crypts and the debris on glass slides.After pooling the appropriate fractions to achieve the greatest percentage of crypt to debris ratio, centrifuge the pooled fractions and aspirate all but the last 50-100 microliters of the supernatants.Keeping the tubes on ice, add 1 milliliter of protein matrix to the pooled fractions, pipetting up and down slowly to prevent the addition of air bubbles.Then, add 50 microliters of the protein matrix/crypt suspension into the middle of each well of 37

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Research

JoVE Journal

Immunology and Infection

13.4K visualizações.  Virginia Maryland College of Veterinary Medicine, Virginia Tech. The overall goal of this procedure is to measure the effects of pathogenic challenge on small intestine organoids with an emphasis on normalization techniques against the total cell number.This method could help answer key questions in the innate as well as mucosal immunology fields by providing a means to investigate host pathogen interactions of bacteria with intestinal epithelial cells in vitro.Essential to this procedure is the method for normalization against the total cell number, a nec...