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.

כל המחקרים נערכו בבדיקה הנחיות קפדניות על פי ועדת טיפול בבעלי חיים מוסדיים השתמש (IACUC) בבית הספר לרפואה של אוניברסיטת הרווארד, אשר עומדת בסטנדרטים וטרינריים שקבעה האגודה האמריקאית למדע בעלי חיים מעבדה (AALAS). 1. הילוכים אופקיים של חיידקים באמצע?...

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אנו מציגים פרוטוקול עבור הבידוד וזרימת אפיון cytometric של לימפוציטים קטנים-מעיים, כולל LPLs, IELs, ואת הלימפוציטים PPS. למעוניינים בהערכה כיצד שינויי החיידקים להשפיע על המערכת החיסונית הקטנה-מעיים, נפרטנו את השלבים הפשוטים המעורבים בהעברה האופקית של אורגניזמים בין עכברי מחס?...

המשימה העיקרית של המעי הדק נחשב לעתים קרובות להיות עיכול וספיגה של חומרים מזינים 1. בעוד תפקוד המטבולי זה חיוני בבירור, המעי הדק יש תפקיד משמעותי לא פחות בהגנת המארח מן המטח המתמיד של אנטיגנים סביבה נמצאים בתוך לומן 2. המעי מפרידה בין העולם החיצוני (למשל., אנטיגנים לומינל) מהסביבה הפנימית של המארח עם שכבת האפיתל כי היא שכבה תא בודד רק עבה. ככזה, המערכת החיסונית הקטנה-מעיים יש את המשימה האדירה של איזון הסף שלה עבור תגובתיות, המאפשר אנטיגנים זרים מן חיידקי דיאטת commensal להיכנס רירי עם מינימאלי, אם בכלל, תגובה חיסונית תוך גובר תגובה חזקה נגד פתוגנים הפולשים &quot;מזיקים&quot; אנטיגנים אחרים. תגובות חיסוניות מוגזמות או לא מתאימות לאנטיגנים אלה יכולים להוביל למחלות פתולוגיים (למשל., Inflammaמחלות מעי טור, סוכרת מסוג I, טרשת נפוצה) ויש להימנע מהם 3-6. בסך הכל, מערכת העיכול הוא חשב לייצג את איבר החיסון הגדול ביותר בגוף, המכיל מעל 70% מכלל התאים מפרישי נוגדן 7. המערכת החיסונית קטן-מעיים מורכב מ -3 תאים עיקריים - שכבת הרירית המיוחדת (LP), השכבה intraepithelial, וטלאים של Peyer (PPS) - שכל אחד מהם מכיל קבוצה ייחודית של לימפוציטים 2. הלימפוציטים LP (LPLs) הם בעיקר תאי TCRαβ + T עם תאים ~ 20% B; לימפוציטים intraepithelial (IELs) מכילים תאי B מעט מאוד עם יותר תאי TCRγδ + T מאשר תאי TCRαβ + T; ו- PPS, שהן איברים הלימפה משני מוטבע בקיר הקטן-מעיים, מכיל ~ 80% תאי B. למרות שכל אחד האזורים האנטומיים הללו יש פונקציות נפרדות מעט ובסיסים אונטולוגית, הם מתפקדים אהאופנה armonized להגן המארח מפני עלבונות פתוגניים. יתר על כן, יש הערכה גוברת כי החיידקים הוא קובע קריטי להתפתחות של מערכת חיסון במעי, עם הכרה גוברת של קשר מאותו המקור בין חיידקים ספציפיים ואת ontogeny של שושלות תאים מסוימות 8,9. יתר על כן, בהתחשב בכך החינוך של מערכת החיסון במעי משפיע התגובה החיסונית לאתרים מרוחקים מבחינה אנטומית (למשל., דלקת פרקים, טרשת נפוצה, דלקת ריאות), זה הפך להיות ברור כי פיתוח של מערכת החיסון במעי רלוונטי יותר תהליכי מחלה מאשר שהוכר בעבר 10 -12. ככזה, עניין בהערכת כמותית מערכת החיסון במעי האריך מעבר אינטראקציות בין מאכסן לפתוגן עכשיו כולל אינטראקציות בין מאכסן commensal ואת בפתוגנזה של מחלות מערכתיות רבות גם כן. בהתחשב בשונות של שיטות קיימות בהבידוד של לימפוציטים מעיים, שיטה כי הוא מותאם במיוחד תשואה, הכדאיות, ועקביות תוך איזון ראוי בין הזמן הנדרש הוא קריטי יותר ויותר. פרוטוקולים שכוללים הדרגתיים Percoll הם זמן העבודה אינטנסיבית יותר, אשר נוטה טעות אנושה, מוביל התשואה משתנית ואת כדאיות 13. בזאת, אנו מספקים פרוטוקול אופטימיזציה עבור הבידוד והאפיון של לימפוציטים מכל 3 תאי החיסון קטנים-המעיים. בנוסף, עניין גובר נתון שינויים מושרי חיידק במערכת החיסונית ברירית, נכלולי צעדים שיכולים לשמש כדי לאפשר את ההעברה האופקית של מיקרואורגניזמים בין העכברים להעריך כיצד שינויים אלה כמותית להשפיע על המערכת החיסונית של המעי.

<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

המעיים - אשר מכילים את המספר הגדול ביותר של תאי מערכת החיסון של כל איבר בגוף - חשופים כל הזמן אנטיגנים זרים, הן חיידקים תזונתיים. בהתחשב שיש לנו הבנה גוברת כי אנטיגנים לומינל אלה לעזור לעצב את התגובה החיסונית וחינוך של תאים חיסוניים בתוך המעי הוא קריטי עבור מספר המחלות מערכתיות, שם כבר עלה עניין באפיון מערכת החיסון במעי. עם זאת, פרוטוקולים רבים שפורסמו הם מפרך זמן רב. אנו מציגים כאן פרוטוקול פשוט עבור הבידוד של לימפוציטים מתוך שכבת הרירית מיוחדת קטנה-מעיים, שכבת intraepithelial, והטלאים של Peyer כי הוא מהיר, לשחזור, ואינו דורש הדרגתי Percoll מייגע. אף על פי הפרוטוקול מתמקד במעי הדק, זה גם מתאים ניתוח של המעי הגס. יתר על כן, אנו מדגישים היבטים מסוימים שעשויות צריך אופטימיזציה נוספת תלויים בככפרו המדעי הספציפיtion. גישה זו גורמת לבידודה של מספר גדול של לימפוציטים קיימא כי לאחר מכן ניתן להשתמש עבור ניתוח תזרים cytometric או אמצעים חלופיים של אפיון.

ניתוח תזרים cytometric של המתלים תא בודד של לימפוציטים קטנים-מעיים אמור להניב אוכלוסייה דיסקרטית של תאים בעלי מאפיינים דומים קדימה פיזור בצד כמו splenocytes (איורים 1 א ו -1 B). הלימפוציטים עשויים להתחיל למות אם הרקמה אינה נשמרת ב 4 ° C. במהלך השלבים הראש?...

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.

אפיון בידוד תזרים Cytometric של Murine קטנים מעיים לימפוציטים

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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Research

JoVE Journal

Immunology and Infection

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

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

בידוד Cytometric זרימה של תאים ראשוניים Murine Type II alveolar אפיתל ללימודים פונקציונליים ומולקולריים

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

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

בידוד של לימפוציטים מעכבר גניטלי חוברות רירית

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

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 including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

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

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

בידוד ובידול של Th17 הנאיביים T מסוג CD4 לימפוציטים

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

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

בידוד תזרים Cytometric ניתוח של תאים חיסוניים ממוח עכבר איסכמי

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

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

בידוד וניתוח תזרים cytometric של תאי הדם ההיקפיים mononuclear הסתננות-Glioma

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

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

בידוד ההפעלה של Murine לימפוציטים

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

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

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

ניתוח בידוד תזרים Cytometric של האדם תאים האנדוצרוויקאלית גמא דלתא T

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

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

לימפוציטים בידוד מן העכבר קטן המעי המערכת החיסונית

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

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

בידוד והתרבות במבחנה של מקרופאגים מכתשי מאתר ואנושי

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

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

העיכול של הכבד מאתר של ניתוח Cytometric זרימת הלימפה תאי אנדותל

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...