אנו רוצים להודות לNIH למימון מענקי R01-AI026328 (קאק) וR01-AI079004 (קאק).

1. הסרת איברי המין <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים את העכבר באמצעות הנחיות מאושרות. הפוך חתך קו אמצע נמוך ולחזור בו עור. </li><li style=";text-align:right;direction:rtl"> מבודד ולהסיר דרכי מין מלאות, מצינור השחלה לפתח נרתיק. </li><li style=";text-align:right;direction:rtl"> פתח את כל דרכי longitudinally עם מספריים. </li><li style=";text-align:right;direction:rtl"> חותך את איבר המין עם מספריים כירורגית עדינים לחתיכות (כ &lt;1 מ&quot;מ) בצלחת פטרי עם RPMI-10/EDTA מלא (ראה את הנוסחא בהמשך) ומערבב את הרקמה בבקבוק 125 מ&quot;ל בטמפרטורת חדר על stirrer מגנטי מוגדרים עבור 15 דקות. הליך זה חוזר על עצמו פעמים ולאחר מכן את supernatant, המכיל האפיתל ופסולת אחרת, נמחק. </li><li style=";text-align:right;direction:rtl"> יוצק תכולת הקנקן דרך מסננת תא (40μm). לשטוף את שאריות EDTA עם מדיום שלם. RPMI/10 מלא: RPMI 500 מ&quot;ל (Gibco), 10% בסרום שור עוברי (FBS), חום מומת-, 5 המ&quot;ל פניצילין / סטרפטומיצין, 5 1M HEPES מ&quot;ל. RPMI-10/EDTA מלא: 10% RPMI (כאמור לעיל) עם E 5μMDTA </li></ol> 2. מעכל את רקמות מערכת מין <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להעביר פיסות רקמה לבקבוק חדש עם RPMI-10/collagenase טרי 20 מ&quot;ל (ראה מתכון בהמשך) ולעכל את הרקמה על 37 מעלות צלזיוס במשך שעה 1 ב stirrer מגנטי מוגדרים נמרצות כדי לעורר את הרקמה. RPMI-10/collagenase מלא: ממש לפני השימוש, collagenase לשקם סוג שמיני (סיגמא; חנות ב -20 ° C, או לפי הוראות היצרן.), להוסיף לRPMI/10 מלא לריכוז סופי של 450-500U/ml. </li><li style=";text-align:right;direction:rtl"> לאחר העיכול הראשון, לאסוף supernatants דרך מסננת תא 100 מיקרומטר, לשמור על תאים על קרח. </li><li style=";text-align:right;direction:rtl"> העברת הרקמות המעוכלות בבקבוק חדש ולחזור על 2.1 פעמים נוספות באמצעות צעד RPMI-10/collagenase מלא טרי עבור סכום כולל של 3 digestions הנפרד. בשלב זה, ללא חלקי רקמה גלויות צריכים להיות בפתרון. </li><li style=";text-align:right;direction:rtl"> ברכה את כל supernatants יחד ממדגם ואוסף דרך מסננת תא 100 מיקרומטר. ספין גאמות למטה. כדורים סלולריים יופרדו באמצעות גרדיאנטים Percoll. </li></ol> 3. הפרדת תאי מערכת מין באמצעות גרדיאנטים Percoll <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את כל ריכוז של Percoll לפני שימוש: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 100% Percoll איזוטוני: התמהיל 9:01 המנייה Percoll (Pharmacia Biotch) לעומת PBS 10x. חומציותו ל 7.4 באמצעות HCL. </li><li style=";text-align:right;direction:rtl"> 40% פתרון Percoll: לערבב 40 מ&quot;ל של Percoll איזוטוני 100% ו 60 מ&quot;ל של RPMI המלא. </li><li style=";text-align:right;direction:rtl"> 70% פתרון Percoll: לערבב 70 מ&quot;ל של Percoll איזוטוני 100% עם 30 מ&quot;ל של RPMI המלא. </li></ul></li><li style=";text-align:right;direction:rtl"> Resuspend כל תא גלולה מהשלב 6 בפתרוני Percoll 40%. נפח שווה של Percoll 70% יהיה בשימוש על מנת להפוך את השיפועים. ישנן שתי דרכים להגדיר צינורות הדרגתיים: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תחת שכבה: להוסיף השעית תא עם Percoll 40% לצינור שיפוע ראשון, ואז בזהירות מתחת לשכבה 70% Percoll. </li><li style=";text-align:right;direction:rtl"> מעל השכבה: להוסיף Percoll 70% לצינור ראשון, ואז בזהירות ובאיטיות לטעון Percoll 40% המכיל את התאים עלעליון. </li></ol></li><li style=";text-align:right;direction:rtl"> דגימות שיפוע תהיינה centrifuged @ 900 XG, בטמפרטורת החדר, במשך 20 דקות עם הבלם כבוי. </li><li style=";text-align:right;direction:rtl"> הלימפוציטים יהיו בשכבת הממשק שבין 40% ושכבות Percoll 70%. זהירות לקצור את שכבת הממשק, לשטוף עם RPMI 01:03 וספין למטה ב740 XG. הלימפוציטים יהיו בתא הכדורי ומוכנים לשימוש נוסף. </li></ol> 4. נציג תוצאות דוגמה לבידוד של לימפוציטים מדרכי מין עכבר וcytometry ניתוח תזרים (FACS) מוצגת באיור 1. דרכי מין הוסרו מכלמידיה muridarum עכבר נדבק intravaginally 7 ימים לאחר הדבקה. שני מסלולי גניטלי היו נקווים יחד כדי שיהיו מספיק לימפוציטים לבדיקה פונקציונלית ופנוטיפ ידי FACS. רקמות גזורות עובדו על ידי העיכול וצעדי בידוד כפי שתואר לעיל. השעית התא הבודדה הייתה מוכתמת לfluorochrome מצומדות השונהנוגדנים חד שבטי כנגד CD3 עכבר, CD4 וCD8. איור 1 מציג מצגת הדוט עלילה של תאי T מסוג CD4 חיוביים לעומת תאי T CD8 חיוביים מגודרים בלימפוציטים מסוג T CD3 חיובי. ההליך שלנו יכול לבחון לימפוציטים שבודדו משטחי אברי מין של מודלים שונים מחלה להעריך תאים חיסוניים מאפיינים שונים ופונקציונליים הפנוטיפי במחלות שונות במודל עכבר. אלה כוללים תאים חיסוניים מגוונים, כגון תאים דנדריטיים, נויטרופילים, מקרופגים NK, NKT, תאי T (תאי T Ag ספציפיים), תאי B וכו 3-10. <img alt="איור 1" src="/files/ftp_upload/4391/4391fig1.jpg" /> האיור 1. ימפוציטים בודדו משטחי אברי מין של עכברים נגועים בכלמידיה muridarum, בשיטה שהוצגה כאן. לאחר הליך ההפרדה המוחלטת, לימפוציטים הוכתמו CD3 fluorochrome-מצומדות, CD4 וCD8. הנתונים של הרביע היו מגודרים בCD3 הלימפוציטים +,מראה מסוג CD4 + לעומת CD8 + ובאחוז התאים מגודרים.

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	<li>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+impact+of+perinatal+immune+development+on+mucosal+homeostasis+and+chronic+inflammation.">The impact of perinatal immune development on mucosal homeostasis and chronic inflammation.</a> Nat. Rev Immunol. 12, 9-23 (2011).</li><li>Centers for Disease Control and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2010 Sexually Transmitted Diseases Surveillance. , (2010).</li><li>Jain, S., Patrick, A. J., Rosenthal, K. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravaginal+infection+with+herpes+simplex+virus+type-2+(HSV-2)+generates+a+functional+effector+memory+T+cell+population+that+persists+in+the+murine+genital+tract.">Intravaginal infection with herpes simplex virus type-2 (HSV-2) generates a functional effector memory T cell population that persists in the murine genital tract.</a> J. Reprod. Immunol. 87, 39-44 (2010).</li><li>Gillgrass, A. E., Tang, V. A., Towarnicki, K. M., Rosenthal, K. 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אין ניגודי האינטרסים הכריזו.

בפרוטוקול זה, הוא הראה כיצד להתכונן לימפוציטים משטחי עכבר אברי מין והוא שולט בקלות. הנקודה הקריטית ביותר של תהליך זה היא לשמור על כדאיויות תא על ידי שמירה על התאים על קרח. תשואת התא משיטה זו תלויה בטכניקה, מיומנות טיפול, מצב המדבק של העכבר (נאיבי או יום אחרי הדבקה), פתוג...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם </td><td> חברה </td><td> מספר קטלוגים </td><td></td></tr><tr><td> Collagenase </td><td> סיגמא מדעי חיים </td><td> C2139-1G </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare ביו מדעים </td><td> 17-0891-01 </td><td></td></tr><tr><td> RPMI1640 </td><td> Gibco ידי טכנולוגיות חיים </td><td> 11875 </td><td></td></tr></tbody></table>

isolation of lymphocytes from mouse genital tract mucosa

משטחים ריריים, כולל בשטחים במערכת העיכול, ואברי מין, ונשימה, מספקים פורטלים של כניסה לפתוגנים, כגון וירוסים וחיידקים 1. Mucosae גם אתרים אינדוקטיביים במארח לייצר חסינות מפני פתוגנים, כגון תיקוני Peyers עובר במעי ורקמות lymphoreticular אף הקשורים בדרך הנשימה. תכונה ייחודית זו מביאה חסינות רירית כשחקן חשוב מאוד במערכת ההגנה של המארח. מחקרים רבים שהתמקדו באתרי רירית מערכת עיכול ומערכת נשימה. עם זאת, יש כבר חקירה קטנה של אתרים ריריים רבייה. אברי המין בדרכי הרירית היא אתר הזיהום העיקרי למחלות המועברות במגע מיני (STD), כולל זיהומים חיידקיים ונגיפיים. מחלות מין הן האחד האתגרים הבריאותיים הקריטיים ביותר העומד בפני עולם כיום. מרכז לבקרת מחלות ומניעתן מעריך כי יש 19,000,000 מדבקת בכל שנה חדשה בארצות הברית. STDs עולה למערכת הבריאות בארה&quot;ב 17 מליארד דולרים בכל שנה 2, ואנשים עוד יותר בעלות השלכות בריאותיות מיידיות וחיים ארוכים. על מנת להתמודד עם אתגר זה, הבנה טובה יותר של חסינות רירית רבייה יש צורך והבידוד לימפוציטים הוא מרכיב חיוני של לימודים אלה. כאן, אנו מציגים שיטת reproducibly לבודד ימפוציטים משטחי Murine מין נשי ללימודים אימונולוגיים כי ניתן לשנות להסתגלות למינים אחרים. השיטה המתוארת להלן מבוססת על עכבר אחד.

Watch this Scientific Journal Video about Isolation of Lymphocytes from Mouse Genital Tract Mucosa at JoVE.com

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

דרך יעילה לבודד את הלימפוציטים מדרכי מין עכבר מתוארת. שיטה זו מנצלת עיכול אנזים והפרדת השיפוע Percoll לאפשר בידוד יעיל. טכניקה זו היא גם להתאמה לשימוש במינים אחרים

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-lymphocytes-from-mouse-genital-tract-mucosa

Research

JoVE Journal

Immunology and Infection

14.3K Views.  University of California, Los Angeles. דרך יעילה לבודד את הלימפוציטים מדרכי מין עכבר מתוארת. שיטה זו מנצלת עיכול אנזים והפרדת השיפוע Percoll לאפשר בידוד יעיל. טכניקה זו היא גם להתאמה לשימוש במינים אחרים

Video: Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

אינדוקציה Transurethral של דלקת בדרכי השתן עכבר

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA בידוד Pseudomonas aeruginosa מיישבים את מערכת העיכול Murine

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

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 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 ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

ניתוח מהיר של סטיות כרומוזום בעכבר לימפוציטים מסוג B על ידי הרשות הפלסטינית-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

פרוטוקול בידוד יעיל עבור B ו- T לימפוציטים משקדי הפלטין אדם

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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.

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 ...

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 ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

לשעבר Vivo דלקת של רירית איברי המין האנושי רקמת הלימפה ונקבה עם וירוס הכשל החיסוני האנושי 1, Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

בידוד של תאים דנדריטים האדם באיברי הרבייה הנקביים ללימודי פנוטיפיות שהיו קיימות בעבר ופונקציונלי

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...