Name: isolation and flow cytometric characterization of murine small intestinal lymphocytes
Uploaded: 2016-05-08T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes at JoVE.com

NKS is supported by NIH award K08 AI108690.

Alle undersøgelser blev udført under streng revision og retningslinjer i henhold til Institutional Animal Care og brug Udvalg (IACUC) ved Harvard Medical School, som opfylder de veterinære standarder fastsat af American Association for Laboratory Animal Science (AALAS). 1. Vandret Transmission Bacteria via Co-hus (valgfri) <ol><li> For at minimere eksogen forurening (især hvis du bruger gnotobiotiske mus), praktiserer aseptisk teknik, mens samle sterile engangssprøjter bure, hjælp mad og vand, der begge ...

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	<li>Cummings, D. E., Overduin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+regulation+of+food+intake.">Gastrointestinal regulation of food intake.</a> J Clin Invest. 117 (1), 13-23 (2007).</li><li>Mowat, A. M., Agace, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+specialization+within+the+intestinal+immune+system.">Regional specialization within the intestinal immune system.</a> Nat Rev Immunol. 14 (10), 667-685 (2014).</li><li>Round, J. L., Mazmanian, S. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+influences+in+inflammatory+bowel+diseases.">Microbial influences in inflammatory bowel diseases.</a> Gastroenterology. 134 (2), 577-594 (2008).</li><li>Tlaskalova-Hogenova, 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=Commensal+bacteria+(normal+microflora),+mucosal+immunity+and+chronic+inflammatory+and+autoimmune+diseases.">Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases.</a> Immunol Lett. 93 (2-3), 97-108 (2004).</li><li>Wen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+and+intestinal+microbiota+in+the+development+of+Type+1+diabetes.">Innate immunity and intestinal microbiota in the development of Type 1 diabetes.</a> Nature. 455 (7216), 1109-1113 (2008).</li><li>Pabst, R., Russell, M. W., Brandtzaeg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+distribution+of+lymphocytes+and+plasma+cells+and+the+role+of+the+gut.">Tissue distribution of lymphocytes and plasma cells and the role of the gut.</a> Trends Immunol. 29 (5), 206-208 (2008).</li><li>Surana, N. K., Kasper, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yin+yang+of+bacterial+polysaccharides:+lessons+learned+from+B.+fragilis+PSA.">The yin yang of bacterial polysaccharides: lessons learned from B. fragilis PSA.</a> Immunol Rev. 245 (1), 13-26 (2012).</li><li>Surana, N. K., Kasper, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+tete-a-tete+between+the+microbiota+and+the+immune+system.">Deciphering the tete-a-tete between the microbiota and the immune system.</a> J Clin Invest. 124 (10), 4197-4203 (2014).</li><li>Gauguet, S., 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=Intestinal+microbiota+of+mice+influences+resistance+to+Staphylococcus+aureus+pneumonia.">Intestinal microbiota of mice influences resistance to Staphylococcus aureus pneumonia.</a> Infect Immun. , (2015).</li><li>Ochoa-Reparaz, J., 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=A+polysaccharide+from+the+human+commensal+Bacteroides+fragilis+protects+against+CNS+demyelinating+disease.">A polysaccharide from the human commensal Bacteroides fragilis protects against CNS demyelinating disease.</a> Mucosal Immunol. 3 (5), 487-495 (2010).</li><li>Wu, H. J., 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=Gut-residing+segmented+filamentous+bacteria+drive+autoimmune+arthritis+via+T+helper+17+cells.">Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells.</a> Immunity. 32 (6), 815-827 (2010).</li><li>Goodyear, A. W., Kumar, A., Dow, S., Ryan, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+murine+small+intestine+leukocyte+isolation+for+global+immune+phenotype+analysis.">Optimization of murine small intestine leukocyte isolation for global immune phenotype analysis.</a> J Immunol Methods. 405, 97-108 (2014).</li><li>Chung, 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=Gut+immune+maturation+depends+on+colonization+with+a+host-specific+microbiota.">Gut immune maturation depends on colonization with a host-specific microbiota.</a> Cell. 149 (7), 1578-1593 (2012).</li><li>Resendiz-Albor, A. A., Esquivel, R., Lopez-Revilla, R., Verdin, L., Moreno-Fierros, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Striking+phenotypic+and+functional+differences+in+lamina+propria+lymphocytes+from+the+large+and+small+intestine+of+mice.">Striking phenotypic and functional differences in lamina propria lymphocytes from the large and small intestine of mice.</a> Life Sci. 76 (24), 2783-2803 (2005).</li><li>Carrasco, 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=Comparison+of+lymphocyte+isolation+methods+for+endoscopic+biopsy+specimens+from+the+colonic+mucosa.">Comparison of lymphocyte isolation methods for endoscopic biopsy specimens from the colonic mucosa.</a> J Immunol Methods. 389 (1-2), 29-37 (2013).</li><li>Van Damme, N., 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=Chemical+agents+and+enzymes+used+for+the+extraction+of+gut+lymphocytes+influence+flow+cytometric+detection+of+T+cell+surface+markers.">Chemical agents and enzymes used for the extraction of gut lymphocytes influence flow cytometric detection of T cell surface markers.</a> J Immunol Methods. 236 (1-2), 27-35 (2000).</li></ol>

Vi præsenterer en protokol til isolering og flowcytometrisk karakterisering af små-tarm lymfocytter, herunder LPLs, IELs, og lymfocytter i PPs. For dem interesseret i at vurdere, hvordan ændringer i mikrobiota påvirker små-tarm immunsystem, vi detaljer de enkle trin i horisontal overførsel af organismer mellem mus huser forskellige microbiotas. Skønt denne protokol fokuserer på tyndtarmen, er proceduren den samme for analyse af tyktarmen, med den eneste forskel er, at der ikke PPs at fjerne (selvom colon patches...

Den vigtigste opgave af tyndtarmen er ofte anset for at være den fordøjelse og optagelse af næringsstoffer 1. Mens dette metaboliske funktion er klart nødvendigt, tyndtarmen har en lige så vigtig rolle i at beskytte værten fra den stadige spærreild af miljømæssige antigener findes inden i hulrummet 2. Tarmkanalen adskiller omverdenen (f.eks., Luminale antigener) fra det indre miljø i værten med et epitel lag, er kun et enkelt cellelag tykt. Som sådan er den lille-tarm immunsystem har den formidable opgave at afbalancere dens tærskel for reaktivitet, hvilket tillader fremmede antigener fra kosten og kommensale mikrober at indtaste slimhinden med minimal, om nogen, immunrespons under montering en robust respons mod invaderende patogener og andre &quot;skadelige&quot; antigener. Overdreven eller upassende immunreaktioner på disse antigener kan føre til patologisk sygdom (f.eks., Inflammatory tarmsygdom, type I-diabetes, dissemineret sklerose) og skal undgås 3-6. Samlet set mavetarmkanalen menes at repræsentere den største immun organ i kroppen, der indeholder over 70% af alle antistof-secernerende celler 7. Den lille-tarm immunsystem består af 3 hovedrum - lamina propria (LP), den intraepithelial lag og Peyerske plaques (PPS) - at hver indeholder en karakteristisk gruppe af lymfocytter 2. LP-lymfocytter (LPLs) er primært TCRαβ + -T-celler med ~ 20% B-celler; intraepithelial lymfocytter (IELs) indeholder meget få B-celler med flere TCRγδ + T-celler end TCRαβ + T-celler; og PPS, som er sekundære lymfoide organer indlejret i mellem-tarmvæggen, indeholder ~ 80% B-celler. Selv om hver af disse anatomiske regioner har lidt forskellige funktioner og ontologiske baser, de fungerer i aharmonized mode at beskytte værten patogene fornærmelser. Endvidere er der voksende forståelse, at mikrobiota er en kritisk determinant for udviklingen af tarmens immunsystem, med stigende anerkendelse af den beslægtede forhold mellem specifikke mikrober og ontogenese af bestemte celleslægter 8,9. Og eftersom uddannelse af tarmens immunsystem påvirker immunresponser i anatomisk fjerne steder (f.eks. Arthritis, dissemineret sklerose, lungebetændelse), er det blevet klart, at udviklingen af det intestinale immunsystem er relevant for flere sygdomsprocesser end tidligere indregnet 10 -12. Som sådan har interesse i kvantitativt at vurdere den intestinale immunsystem forlænges ud værtspatogene interaktioner nu inkluderer værts-kommensal interaktioner og patogenesen af ​​mange systemiske sygdomme samt. Omfatter variationer nuværende metoder iisolering af intestinale lymfocytter, en metode, der er optimeret til udbytte, levedygtighed, og konsistens samtidig med at balancere den nødvendige tid i stigende grad kritisk. Protokoller, der involverer Percoll gradienter er tids- og arbejdskrævende og potentielt mere tilbøjelige til menneskelige fejl, der fører til variabel rente og levedygtighed 13. Heri giver vi en optimeret protokol til isolering og karakterisering af lymfocytter fra alle 3 små-tarm immune rum. Derudover gives stigende interesse for mikrobe-inducerede ændringer i det mucosale immunsystem, inkluderer vi trin, der kan anvendes til at give mulighed for horisontal overførsel af mikroorganismer mellem mus til at vurdere, hvordan disse ændringer kvantitativt påvirker intestinale immunsystem.

<table>
	<tbody>
		<tr>
			<td>Sterile Gloves</td>
			<td>Kimberly-Clark</td>
			<td>555092</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile mouse cage</td>
			<td>Innovive</td>
			<td>MS2-AD</td>
			<td>contains lid, cage bottom, and alpha-dri bedding</td>
		</tr>
		<tr>
			<td>metal feeder</td>
			<td>Innovive</td>
			<td>M-FEED</td>
			<td></td>
		</tr>
		<tr>
			<td>water bottle</td>
			<td>Innovive</td>
			<td>M-WB-300</td>
			<td></td>
		</tr>
		<tr>
			<td>card holder</td>
			<td>Innovive</td>
			<td>CRD-HLD-H</td>
			<td></td>
		</tr>
		<tr>
			<td>autoclavable rodent chow (NIH-31M)</td>
			<td>Zeigler</td>
			<td>4131207530</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>Ambion</td>
			<td>AM9262</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>GemBio</td>
			<td>100-510</td>
			<td></td>
		</tr>
		<tr>
			<td>dispase II</td>
			<td>Invitrogen</td>
			<td>17105-041</td>
			<td>the concentration in the protocol is based on an activity level of 1.878 U/mg</td>
		</tr>
		<tr>
			<td>collagenase, type II&#160;</td>
			<td>Invitrogen</td>
			<td>17101-015</td>
			<td>the concentration in the protocol is based on an activity level of 245 U/mg</td>
		</tr>
		<tr>
			<td>dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>feeding needle (18 G, 2&#34; length)</td>
			<td>Roboz</td>
			<td>FN-7905</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml syringe</td>
			<td>BD</td>
			<td>305482</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel (15 blade)</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
		</tr>
		<tr>
			<td>curved forceps</td>
			<td>Roboz</td>
			<td>RS-5211</td>
			<td></td>
		</tr>
		<tr>
			<td>straight forceps</td>
			<td>Roboz</td>
			<td>RS-5132</td>
			<td></td>
		</tr>
		<tr>
			<td>multi-purpose cups, 120 ml</td>
			<td>VWR</td>
			<td>89009-662</td>
			<td></td>
		</tr>
		<tr>
			<td>stir bar</td>
			<td>VWR</td>
			<td>58949-062</td>
			<td></td>
		</tr>
		<tr>
			<td>multi-position stir plate, 9-position</td>
			<td>VWR</td>
			<td>12621-048</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel conical strainer, 3 inch&#160;</td>
			<td>RSVP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml tube</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate, round-bottom</td>
			<td>Corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD16/32 (Fc block)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td></td>
		</tr>
		<tr>
			<td>(optional) fixable viability dye eFluor 780</td>
			<td>eBiosciences</td>
			<td>65-0865-18</td>
			<td></td>
		</tr>
		<tr>
			<td>10% formalin, neutral buffered</td>
			<td>Thermo Scientific</td>
			<td>5725</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric characterization of murine small intestinal lymphocytes

Tarmene - der indeholder det største antal af immune celler i alle organer i kroppen - er konstant udsat for fremmede antigener, både mikrobiel og kosten. Givet en stigende forståelse af, at disse luminale antigener at forme immunresponset og at uddannelse af immune celler i tarmen er kritisk for en række systemiske sygdomme, har der været en stigende interesse for at karakterisere den intestinale immunsystem. Men mange publicerede protokoller er besværlig og tidskrævende. Vi præsenterer her en forenklet protokol til isolering af lymfocytter fra den lille-tarm lamina propria, intraepithelial lag, og Peyerske plaques, som er hurtig, reproducerbar, og kræver ikke møjsommelige Percoll-gradienter. Selvom protokollen fokuserer på tyndtarmen, er det også egnet til analyse af tyktarmen. Desuden fremhæver vi nogle aspekter, der kan have brug for yderligere optimering afhængigt af den specifikke videnskabelige spørgstion. Denne fremgangsmåde resulterer i isoleringen af ​​et stort antal levedygtige lymfocytter, som efterfølgende kan anvendes til flowcytometrisk analyse eller alternative midler til karakterisering.

Flow-cytometri af enkeltcellesuspensioner af små-tarm lymfocytter skulle give en diskret population af celler, der har lignende fremad og sidespredning egenskaber som splenocytter (figur 1A og 1B). Lymfocytterne kan begynde at dø, hvis vævet ikke holdes ved 4 ° C under de indledende stadier af isolering, hvilket resulterer i lymfocytpopulationen har en nedre forward scatter og være mere vanskeligt at adskille fra andre epitel- og døde celler (figur 1C)</str...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes at JoVE.com

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Isolering og Flowcytometrisk karakterisering af murine Lille Intestinal Lymfocytter

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

The overall goal of this procedure is to isolate single cell populations from different small intestinal compartments that can subsequently be used for flow cytometric analysis or an alternate means of characterization. This method can help answer key questions in mucosal immunology such as how the microbiota impacts the development of the intestinal immune system. The main advantages of this technique are that they are rapid, reproducible and don't require laborious percoll gradients.To begin this procedure, place the mouse dorsal side down and spray the abdomen with 70 percent ethanol, perform a laparotomy by sequentially cutting the skin and then peritoneal fascia along the ventral midline from the pubic symphysis to the xiphoid process, thus exposing the peritoneal cavity. Next, separate the small intestine from the stomach by transecting the pyloric sphincter. Gently remove the small intestine from the peritoneum and tease away the mesenteric fat.To fully remove the small intestine, make a second cut at the ileocecal junction. Place the isolated small intestine into four degree Celsius RPMI medium containing 10 percent FBS to maximize the cell viability. Gently remove large pieces of fat using curved forceps and use caution to avoid tearing the intestinal tissue itself during the process.To remove the intestinal contents, cannulate the proximal end of the small intestine with an 18 gauge feeding needle affixed to a syringe and gently flush the intestine with 15 to 20 milliliters of cold PBS. Use scissors to excise Peyer's Patches which are located on the antimesenteric side of the small intestine and appear as a multilobulated white mass. Then, place them into cold RPMI medium containing five percent FBS.Next, cut the small intestine into three to four inch segments, remove the residual fat by rolling each small intestinal segment on a paper towel moistened with RPMI and use a dull scalpel to tease the fat away from the tissue. Paying strict attention to the complete removal of mesenteric fat is critical to ensuring the viability of lymphocytes as even minute pieces of fat can cause cells to die. After that, turn the tissue inside out by cannulating the intestinal segments with curved forceps and grasping the distal end of the tissue, then use a pair of straight forceps to gently remove the tissue segment from the proximal end, resulting in the tissue being inverted.Place the tissue segments in a cup containing 30 milliliters of extraction media and a stir bar. Secure the lid on the cap and stir for 15 minutes at 37 degrees Celsius. Stirring should be vigorous, but not turbulent.After 15 minutes, use a steel strainer to separate the tissue pieces from the epithelium containing supernatant which should appear cloudy. Manually agitate the tissue pieces in RPMI medium to wash away the residual extraction media. Then, place the tissue on a dry paper towel, and flip it several times to facilitate the removal of residual mucous that was not liberated by the extraction medium.Subsequently, place the tissue fragments in a one point five milliliter tube with 600 microliters of digestion medium. Mince the tissue until the pieces no longer stick to the scissors and the solution appears homogenous. This step is critical to ensure complete enzymatic digestion of the tissue.Mincing the tissue until the pieces no longer stick to the scissors and the solution appears homogeneous are critical to ensuring cellular yield as well as ensuring reproducibility of results. Add the minced small intestine to a cup containing 25 milliliters of digestion media and stir for 30 minutes at 37 degrees Celsius. Halfway through the digestion, pipe it up and down with a serological pipette to help break down any large chunks of tissue.Next, filter the digestive tissue through a 100 micrometer cell strainer into a 15 milliliter tube. Rinse the strainer with 20 milliliters of RPMI medium containing 10 percent FBS. Afterward, centrifuge the filtered solution for 10 minutes at four degrees Celsius.Carefully decant the supernatant and re-suspend the pellet in one milliliter of RPMI medium containing 10 percent FBS. Filter the re-suspended cells through a 40 micrometer cell strainer into a 15 milliliter tube. Rinse the strainer with 20 milliliters of RPMI containing 10 percent FBS.Subsequently, centrifuge the filtered solution for 10 minutes at four degrees Celsius. Carefully decant the supernatant and re-suspend the pellet in one milliliter of RPMI medium, containing two percent FBS. In this procedure, transfer the excised Peyer's Patches to a cup with 25 milliliters of digestion media and a stir bar.Secure the lid and spin for 10 minutes at 37 degrees Celsius. Afterward, filter the digested Peyer's Patches through a 40 micrometer cell strainer into a 15 milliliter tube. If any clumps remain, press them through the strainer using the flat end of the plunger from a one milliliter syringe.Then, rinse the strainer with 10 milliliters of RPMI medium, containing 10 percent FBS. Centrifuge the filtered solution for 10 minutes at four degrees Celsius. Then, carefully decant the supernatant and re-suspend the pellet in one milliliter of RPMI medium containing two percent FBS.Flow cytometric analysis of single cell suspensions of small intestinal lymphocytes, should yield a discreet population of cells that have similar forward and side scatter characteristics as splenocytes. The lymphocytes may began to die if the tissue is not maintained at four degrees Celsius during the initial stages of the isolation, resulting in the lymphocyte population having a lower forward scatter and being more difficult to separate from other epithelial and dead cells. Moreover, if the mesenteric fat is not completely removed from the small intestinal tissue, there will be a virtually complete loss of lymphocytes.Typically, an 80 percent viability for small intestinal LP lymphocytes is obtained. Once mastered, one can process four mice and have single cell suspensions ready for staining in less than four hours. During this procedure, it's important to completely remove all mesenteric fat and to mince the tissue well to ensure complete digestion.Following this procedure, single cell suspensions can be used for purposes other than flow cytometry, such as en vitro experiments, gene expression analysis or adoptive transfer to further characterize the function of these cells. After its development, this technique paved the way for researchers in mucosal immunology to explore the ontogeny and functional implications of the intestinal immune system in mice. After watching this video, you should have a good understanding of how to isolate single cell suspensions from different small intestinal compartments, by cleaning the mesenteric fat, extracting the epithelial layer, and digesting the tissue with collagenase.

Research

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Immunology and Infection

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Flowcytometrisk Isolering af primære murine type II alveolære epitelceller for Funktionelle og molekylære studier

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Isolering af lymfocytter fra Mouse Genital Tract mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

Isolering og Th17 Differentiering af Naïve CD4 T-lymfocytter

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Isolering og flowcytometrisk analyse af immunceller fra iskæmisk Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Isolering og flowcytometrianalyse gliom-infiltrerende perifere mononukleære blodceller

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Activation of Murine Lymphocytes

Isolering og aktivering af murine lymfocytter

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometri af partikelbundet Bet v 1 Allergen i PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

Isolering og flowcytometrisk analyse for Human endocervikale Gamma Delta T-celler

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

Isolere lymfocytter fra mus Small Intestinal immunsystemet

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation og In Vitro kultur af Murine og menneskelige alveolær makrofager

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...