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.

All studies were conducted under strict review and guidelines according to the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School, which meets the veterinary standards set by the American Association for Laboratory Animal Science (AALAS).
1. Horizontal Transmission of Bacteria via Co-housing (Optional)&#160;
<ol>
	<li>To minimize exogenous contamination (particularly if using gnotobiotic mice), practice aseptic technique while assembling sterile disposable cages, using food and water t...

<ol>
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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>

We present a protocol for the isolation and flow cytometric characterization of small-intestinal lymphocytes, including the LPLs, IELs, and lymphocytes in the PPs. For those interested in evaluating how changes in the microbiota affect the small-intestinal immune system, we detail the straightforward steps involved in the horizontal transmission of organisms between mice harboring different microbiotas. Although this protocol focuses on the small intestine, the procedure is the same for analysis of the large intestine, w...

The principal task of the small intestine is often considered to be the digestion and absorption of nutrients1. While this metabolic function is clearly essential, the small intestine has an equally significant role in protecting the host from the continual barrage of environmental antigens found within the lumen2. The intestinal tract separates the outside world (e.g., luminal antigens) from the internal environment of the host with an epithelial layer that is only a single cell layer thick. As such, the small-intestinal immune system has the formidable task of balancing its threshold for reactivity, allowing foreign antigens from the diet and commensal microbes to enter the mucosa with minimal, if any, immune response while mounting a robust response against invading pathogens and other &#34;harmful&#34; antigens. Excessive or inappropriate immune responses to these antigens can lead to pathologic disease (e.g., inflammatory bowel disease, type I diabetes, multiple sclerosis) and must be avoided3-6.
Overall, the gastrointestinal tract is thought to represent the largest immune organ in the body, containing over 70% of all antibody-secreting cells7. The small-intestinal immune system is comprised of 3 main compartments&#160;&#8212;&#160;the lamina propria (LP), the intraepithelial layer, and Peyer&#39;s patches (PPs) &#8212;&#160;that each contains a distinctive group of lymphocytes2. The LP lymphocytes (LPLs) are primarily TCR&#945;&#946;+ T cells with ~20% B cells; intraepithelial lymphocytes (IELs) contain very few B cells with more TCR&#947;&#948;+ T cells than TCR&#945;&#946;+ T cells; and PPs, which are secondary lymphoid organs embedded in the small-intestinal wall, contain ~80% B cells. Although each of these anatomical regions has slightly distinct functions and ontological bases, they function in a harmonized fashion to protect the host from pathogenic insults.
Furthermore, there is growing appreciation that the microbiota is a critical determinant for the development of the intestinal immune system, with increasing recognition of the cognate relationship between specific microbes and the ontogeny of particular cell lineages8,9. Moreover, given that education of the intestinal immune system affects immune responses in anatomically distant sites (e.g., arthritis, multiple sclerosis, pneumonia), it has become clear that development of the intestinal immune system is relevant to more disease processes than previously recognized10-12. As such, interest in quantitatively assessing the intestinal immune system has extended beyond host-pathogen interactions to now include host-commensal interactions and the pathogenesis of many systemic diseases as well.
Given the variability of current methods in the isolation of intestinal lymphocytes, a method that is optimized for yield, viability, and consistency while balancing the time required is increasingly critical. Protocols that involve Percoll gradients are time and labor intensive and potentially more prone to human error, leading to variable yield and viability13. Herein, we provide an optimized protocol for the isolation and characterization of lymphocytes from all 3 small-intestinal immune compartments. Additionally, given increasing interest in microbe-induced alterations in the mucosal immune system, we include steps that can be used to allow for the horizontal transmission of microorganisms between mice to assess how these changes quantitatively affect the intestinal immune system.

<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

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 microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

Flow cytometric analysis of single cell suspensions of small-intestinal lymphocytes should yield a discrete population of cells that have similar forward and side scatter characteristics as splenocytes (Figures 1A and 1B). The lymphocytes may begin to die if the tissue is not maintained at 4 &#176;C 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 an...

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.

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.

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