עבודה זו נתמכה בחלקה על ידי NIH מענק AI069358 וקרן מחקר BloodCenter.

<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפני שמתחילים את התהליך, את הפריטים הבאים צריכים להיות מוכנים אסף: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קרח </li><li style=";text-align:right;direction:rtl"> מזרק 5 מ&quot;ל עם מחט 27g </li><li style=";text-align:right;direction:rtl"> מזרק 5 מ&quot;ל עם מחט 25 גרם </li><li style=";text-align:right;direction:rtl"> בלוק קלקר וסיכות להרכבה על העכבר </li><li style=";text-align:right;direction:rtl"> מגש שעליו לחסום הרכבה ניתן למקם </li><li style=";text-align:right;direction:rtl"> מספריים ומלקחיים </li><li style=";text-align:right;direction:rtl"> אוסף צינורות </li><li style=";text-align:right;direction:rtl"> 70% אתנול </li><li style=";text-align:right;direction:rtl"> PBS עם 3% עגל בסרום עוברי (FCS) (Pre צונן ושמרה על הקרח) </li></ul></li><li style=";text-align:right;direction:rtl"> להרדים את העכבר, לרסס אותו עם 70% אתנול ו לעלות אותו על בלוק קלקר על גבו. </li><li style=";text-align:right;direction:rtl"> בעזרת מספריים מלקחיים לחתוך את העור החיצונית של הצפק בעדינות למשוך אותו לאחור כדי לחשוף את העור הפנימית רירית חלל הצפק. </li><li style=";text-align:right;direction:rtl"> הזרק 5 מ&quot;ל של קרח קר PBS (עם FCS 3%) לתוך חלל הצפק באמצעות מחט 27g. דחוף את המחט לאט הצפק נזהרים שלא לנקב כל האיברים. </li><li style=";text-align:right;direction:rtl"> לאחר ההזרקה, בעדינות לעסות את הצפק כדי לסלק את כל התאים מחוברים אל הפתרון PBS. </li><li style=";text-align:right;direction:rtl"> הוסף 25 גרם, שפוע המחט, מצורף מזרק 5 מ&quot;ל ב הצפק לאסוף את הנוזל תוך כדי תנועה את קצה המחט בעדינות, כדי למנוע סתימת על ידי רקמת שומן או איברים אחרים. כמו איסוף הנוזלים כמה שניתן להפקיד את ההשעיה תא שנאספו צינורות שמרה על הקרח לאחר הסרת המחט של המזרק. אופציונלי: חזור על שלב 4-6 </li><li style=";text-align:right;direction:rtl"> עושים חתך בעור הפנימי של הצפק ובעוד מרים את העור עם מלקחיים להשתמש פיפטה פסטר מפלסטיק כדי לאסוף את הנוזלים הנותרים מחלל. </li><li style=";text-align:right;direction:rtl"> אם בשלב 6 או 7 זיהום דם גלוי מזוהה אז המדגם מזוהמים צריך להיות מושלך. </li><li style=";text-align:right;direction:rtl"> ספין על השעיית התא נאספים 1500 סל&quot;ד במשך 8 דקות, לבטל את supernatant ו resuspend התאים התקשורת הרצוי או PBS עבור הספירה. </li></ol> פרוטוקול אלטרנטיבית להשיג thioglycollate שהושרו מקרופאגים: שיטה זו משמשת כדי להשיג תשואה גבוהה יותר של מקרופאגים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הזרק 5 מ&quot;ל של 3% (w / v) ברואר thioglycollate בינוני (7) לתוך חלל הצפק של עכבר אחד. </li><li style=";text-align:right;direction:rtl"> המתן 3-5 ימים, המשך לשלב 1 לעיל לאיסוף של התאים. </li></ol> לעומת הגישה nonelicited, כ 10 פעמים יותר מקרופאגים ניתן לאסוף. החשש היחיד הוא מתקבל על ידי מקרופאגים הליך זה שונה בכמה מאפיינים פיזיולוגיים שלהם. נציג התוצאות: מתוך עכבר unmanipulated, 5-10000000 התאים חלל הצפק ניתן להשיג עם פרוטוקול בידוד טוב. בין כל תאים חיים, 50-60% הם תאים B, מקרופאגים ~ 30% ו 5-10% הם תאים T (איור 1). <img src="/files/ftp_upload/1488/1488fig1.jpg" alt="איור 1" /> באיור 1. הפנוטיפ של תאים בודדים מתוך חלל הצפק. הבידוד בעקבות התאים חלל הצפק הם הוכתמו עכבר אנטי TCRβ-FITC, B220-PE-טקסס אדום, כחול פסיפיק CD11b, CD23-PE-Cy7 CD5 ו-APC. מגרשים תזרים נציג cytometric להראות אחוזים של תאים B ו-T (א), מקרופאגים (ב), B1 ו-B2 תאים (ג) ו B1a ו B1b תאים (ד).

<ol>
	<li>Zhang, X., Goncalves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+characterization+of+murine+macrophages.">The isolation and characterization of murine macrophages.</a> Current Protocols in Immunology. , (2008).</li><li>Boes, M., Prodeus, A. P., Schmidt, T., Carroll, M. C., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+of+natural+immunoglobulin+M+in+immediate+defense+against+systemic+bacterial+infection.">A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection.</a> J. Exp. Med. 188, 2381-2386 (1998).</li><li>Mc Daniel, L. S., Benjamin, W. H., Forman, C., Briles, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+clearance+by+anti-phosphocholine+antibodies+as+a+mechanism+of+protection+in+experimental+pneumococcal+bacteremia.">Blood clearance by anti-phosphocholine antibodies as a mechanism of protection in experimental pneumococcal bacteremia.</a> J. Immunol. 133, 3308-3312 (1984).</li><li>Paciorkowski, N., Porte, P., Shultz, L. D., Rajan, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B1+B+lymphocytes+play+a+critical+role+in+host+protection+against+lymphatic+filarial+parasites.">B1 B lymphocytes play a critical role in host protection against lymphatic filarial parasites.</a> J. Exp. Med. 191, 731-736 (2000).</li><li>Mercolino, T. J., Arnold, L. W., Hawkins, L. A., Haughton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+mouse+peritoneum+contains+a+large+population+of+Ly-1++(CD5)+B+cells+that+recognize+phosphatidyl+choline:+relationship+to+cells+that+secrete+hemolytic+antibody+specific+for+autologous+erythrocytes.">Normal mouse peritoneum contains a large population of Ly-1+ (CD5) B cells that recognize phosphatidyl choline: relationship to cells that secrete hemolytic antibody specific for autologous erythrocytes.</a> J. Exp. Med. 168, 687-698 (1988).</li><li>O'Garra, A., Chang, R., Go, N., Hastings, R., Haughton, G., Howard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ly-1+B+(B-1)+cells+are+the+main+source+of+B+cell+derived+interleukin+10.">Ly-1 B (B-1) cells are the main source of B cell derived interleukin 10.</a> Eur. J. Immunol. 22, 711-717 (1992).</li><li>Hoover, D. L., Nacy, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+to+kill+Leishmania+tropica:+Defective+intracellular+killing+of+amastigotes+by+macrophages+elicited+with+sterile+inflammatory+agents.">Macrophage activation to kill Leishmania tropica: Defective intracellular killing of amastigotes by macrophages elicited with sterile inflammatory agents.</a> J. Immunol. 132, 1487-1491 (1984).</li><li>Austin, P. E., McCulloch, E. A., Till, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+factor+in+L-cell+conditioned+medium+capable+of+stimulating+colony+formation+by+mouse+marrow+cells+in+culture.">Characterization of the factor in L-cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture.</a> J. Cell Physiol. 77, 121-134 (1971).</li><li>Stanley, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macrophage+colony-stimulating+factor,+CSF-1.">The macrophage colony-stimulating factor, CSF-1.</a> Methods Enzymol. 116, 564-587 (1985).</li><li>Baumgarth, N., Chen, J., Herman, O. C., Jager, G. C., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+B-1+and+B-2+cells+in+immune+protection+from+influenza+virus+infection.">The role of B-1 and B-2 cells in immune protection from influenza virus infection.</a> Curr. Top. Microbiol. Immunol. 252, 163-169 (2000).</li><li>Baumgarth, N., Herman, O. C., Jager, G. C., Brown, L. E., Herzenberg, L. A., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-1+and+B-2+cell-derived+immunoglobulin+M+antibodies+are+nonredundant+components+of+the+protective+response+to+influenza+virus+infection.">B-1 and B-2 cell-derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection.</a> J. Exp. Med. 192, 271-280 (2000).</li><li>Berland, R., Wortis, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+functions+of+B-1+cells+with+notes+on+the+role+of+CD5.">Origins and functions of B-1 cells with notes on the role of CD5.</a> Annu. Rev. Immunol. 20, 253-300 (2002).</li></ol>

בידוד של תאים חלל הצפק היא טכניקה חשובה למחקר של תאים חיסוניים שונים, בעיקר מאקרופאגים ספציפי תת תא B. אמנם זה תהליך פשוט, יש כמה שלבים קריטיים מעורב. נוכחות של זיהום בדם במדגם שנאסף יש להימנע להשיג תא טהור חלל הצפק האוכלוסייה. המתת חסד על ידי נקע בצוואר הרחם צריך להיעשות בזהירות כדי למנוע זיהום דם בתוך חלל הבטן. לחילופין CO 2 יכול לשמש. בנוסף, במהלך הטיפול בהליך תקין יש לנקוט כדי למנוע ניקוב השלפוחית ​​או לאיברים אחרים בתוך חלל הבטן. שחזור ביותר של הנוזל מוזרק חשוב גם תשואה התא. הליך זה נעשה שימוש נרחב ללמוד ביולוגיה macrophage מאז מספרים מתונה של תושב, מקרופאגים unstimulated יכול בקלות להיות המתקבל חלל הצפק, בניגוד המשימה המפרכת להבדיל ובתאים מיאלואידית לתוך מקרופאגים בוגרת במבחנה, באמצעות המושבה macrophage גורם מגרה (8 , 9). התשואה macrophage מתוך חלל הצפק ניתן לשפר על ידי שימוש בשיטת התמחרות thioglycollate, למרות השימוש thioglycollate עשוי לשנות את המאפיינים הפיזיולוגיים של מקרופאגים (7). בתאי B הם חלק חשוב של המערכת החיסונית שלנו משחקת תפקיד מכריע הן חסינות מולדת ובעלי כושר הסתגלות. בין תאים שונים subpopulations B, תאים B1 מהווים קבוצת משנה ייחודית של תאים B המעורבים חסינות אוטואימוניות מולדת, תקנה החיסונית. בתאים B1 ממוקמים בעיקר חלל הצפק הם חידוש עצמי. הם אחד היצרנים העיקריים של IgM טבעי המספק את השורה הראשונה של הגנה כנגד מספר וירוסים וחיידקים (10-12). בתאים B1 הם גם מקור עיקרי של IL-10 (6), המהווה ציטוקינים חשוב המעורב בויסות החיסון. למרות מספר מחקרים כבר נרדף עם התאים B1, עדיין יש מרחב לצורך הערכה נוספת של פונקציות מנוגדות שלהם, במיוחד תפקיד הפיקוח שלהם. חלל הצפק תא שיטת בידוד מספק הזדמנות ייחודית ללמוד את הפונקציות של תאים B1.

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<td>Thioglycollate Medium, Brewer Modified</td>
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isolation of mouse peritoneal cavity cells

חלל הצפק הוא קרום חלל הבטן, כפות מלאות נוזל של יונקים, אשר מכיל את הכבד, הטחול, רוב דרכי העיכול ואת הקרביים האחרים. זה מכיל מספר תאים חיסוניים כולל מקרופאגים, תאים B ותאי T. נוכחות מספר גבוה של מקרופאגים נאיבי חלל הצפק שהופך אותו לאתר מועדף לאיסוף נאיבי תושב מקרופאגים ברקמה (1). חלל הצפק חשוב גם במחקר בתאי B בגלל נוכחות של תת קבוצה ייחודית חלל הצפק תושב B לתא המכונה התאים B1 בנוסף התאים B2 קונבנציונאלי. בתאים B1 מחולקים לתאים B1a ו B1b, אשר יכול להיות מכובדת על ידי ביטוי את פני השטח של CD11b ו CD5. בתאים B1 הם מקור חשוב של IgM טבעי מתן הגנה מוקדם ממגוון רחב של פתוגנים (2-4). תאים אלה הם autoreactive בטבע (5), אבל איך הם נשלטים על מנת למנוע אוטואימוניות עדיין אינה מובנת לחלוטין. להיפך, CD5<sup&gt; +</sup&gt; B1a תאים בעלי מאפיינים מסוימים הרגולציה מכוח יכולתם לייצר IL-10 (6). לכן, חלל הצפק בתאים B1 הם אוכלוסיית תאים מעניין ללמוד כי תפקוד מגוונים שלהם unaddressed שאלות רבות הקשורות התפתחותם ורגולציה. הבידוד של חלל הצפק תאים תושב החיסונית היא בעייתית בשל היעדר מבנה מוגדר בתוך חלל הצפק. פרוטוקול שלנו יתאר הליך לקבלת תאים חיסוניים קיימא מתוך חלל הצפק של עכברים, אשר לאחר מכן ניתן להשתמש לצורך ניתוח פנוטיפי ידי cytometry זרימה ועל מבחני ביוכימיים שונים החיסונית.

Watch this Scientific Journal Video about Isolation of Mouse Peritoneal Cavity Cells at JoVE.com

Isolation of Mouse Peritoneal Cavity Cells

חלל הצפק ביונקים מכילה אוכלוסיות תאים שונות החיסונית חיוני תגובות חיסוניות מולדות. שיטת בידוד יעיל נדרש ניתוח ביוכימי ותפקודי של תאים אלה. כאן אנו מספקים שיטה מקיפה עבור בידוד של תאים חלל הצפק בתוך העכבר.

בידוד של תאים עכבר פריטוניאלית חלל

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

isolation-of-mouse-peritoneal-cavity-cells

Research

JoVE Journal

Immunology and Infection

142.7K Views.  Blood Research Institute. חלל הצפק ביונקים מכילה אוכלוסיות תאים שונות החיסונית חיוני תגובות חיסוניות מולדות. שיטת בידוד יעיל נדרש ניתוח ביוכימי ותפקודי של תאים אלה. כאן אנו מספקים שיטה מקיפה עבור בידוד של תאים חלל הצפק בתוך העכבר.

Video: Isolation of Mouse Peritoneal Cavity Cells

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

בידוד של תאים חיסוניים פונקציונלית לב

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

בידוד של ריאות תאים דנדריטים עכבר

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

בידוד ואיפיון של תאים דנדריטים ומקרופגים מהמעי עכבר

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

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 of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

בידוד של תאים חיסוני רקמת שומן

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

בידוד של הצפק Murine המקרופאגים לבצע ניתוח ביטוי גנים בעת ביצוע כמו אגרה רצפטורים הגירוי

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

תאי בידוד של תאי Sertoli ו Peritubular מן חולדת האשכים

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

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 ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

שימוש משופרת פלורסצנטיות ירוק חלבון-הבעת Escherichia Coli כדי להעריך את העכבר מקרופאג הצפק Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

Brush cells are rare cholinergic chemosensory epithelial cells found in the na&#239;ve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

בידוד והערכה כמותית של תאי המברשת מקנה הנשימה של העכבר

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...