מבצע התורם הינד ו-Harvest לימב <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> במשך ההליך האופרטיבי להגדיר סטרילית של microsurgical מכשירים מנוצל. המנתח צריך ללבוש לבוש הולם, כולל כפפות סטריליות, אשר צריך להיות שונה לפני תחילת ההליך הכירורגי. </li><li style=";text-align:right;direction:rtl"> חולדה הוא הרדים עם זריקה intraperitoneal של pentobarbital (IP 50mg/kg) והניח פרקדן על שדה הניתוח מחוממת, המאפשר טמפרטורת גוף יציבה החיה לאורך כל ההליך הכירורגי. </li><li style=";text-align:right;direction:rtl"> לפני חתך עור הגישה של המבצע הוא מחוטא שלוש פעמים כל אחד עם שני, חיטוי ואלכוהול. כדי למנוע את קרניות חיות מהתייבשות, העיניים משומנים עם משחת עיניים (Vidisic) לפני הניתוח. </li><li style=";text-align:right;direction:rtl"> החתך בעור הוא עשה פני במפשעה ימין בערך ברמה של הרצועה מפשעתי. כלי הירך הם גזור חופשי עם הפרעה דו כויה ו דיסקציה קהה; ענפים עמוק הם ligated עם משי 8-0. </li><li style=";text-align:right;direction:rtl"> 300μl (50 IU) של הפרין מוזרקים דרך הווריד הפין עבור אנטי קרישה. </li><li style=";text-align:right;direction:rtl"> כלי עצם הירך הם ligated ברמה של הרצועה מפשעתי וחותכים דיסטלי אל המיתר. </li><li style=";text-align:right;direction:rtl"> איבר האחוריות היא הסמיקה קר כקרח (4 ° C) פתרון HTK (5 מ&quot;ל) דרך עורק הירך מעל 5 דקות. </li><li style=";text-align:right;direction:rtl"> כל קבוצות השרירים נחלקים באופן חד עם מספריים osteotomy מבוצע עם חשמלי מסתובב ראה ברמה של בשליש דיסטלי של עצם הירך. </li><li style=";text-align:right;direction:rtl"> איבר שנקטפו הוא שקוע אז 20mL של פתרון קרים כקרח HTK לשימור בקירור עד להשתלה. </li></ol> נמען מבצע והשתלות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חולדה הוא הרדים עם זריקה intraperitoneal של פרקדן pentobarbital והניחה על שדה הניתוח. </li><li style=";text-align:right;direction:rtl"> החתך בעור הוא עשה פני במפשעה ימין בערך ברמה של הרצועה מפשעתי. כלי הירך הם גזור חופשי עם הפרעה דו כויה ו דיסקציה קהה. </li><li style=";text-align:right;direction:rtl"> כלי עצם הירך הם ligated כמו דיסטלי ככל האפשר, אך בקרבה ההמראה כלי ברום הבטן. מהדק דם ממוקמים ברמה של הרצועה מפשעתי על העורק לווריד. </li><li style=";text-align:right;direction:rtl"> שרוול פוליאתילן הוא עצר העורק (קוטר בתוך השרוול: 0.912, מ&quot;מ השרוול קיר עובי: 0.010 מ&quot;מ, אורך השרוול: 2.0 מ&quot;מ) וריד (בקוטר בתוך השרוול: 1.151 מ&quot;מ; השרוול קיר עובי: 0.010 מ&quot;מ, אורך השרוול : 2.0 מ&quot;מ). בקצה לומן כלי נמשך אז עם המרחיב כלי ואת עורק וריד הירך הם everted על הגוף של השרוול (כ 2 מ&quot;מ) ו קבוע עם ליגטורה 8-0 ההיקפי משי. </li><li style=";text-align:right;direction:rtl"> כדי לפנות מקום איבר איבר התורם למקבל יוסר עם דיסקציה חריפה של קבוצות שרירים osteotomy מבוצעת אמצע הירך. </li><li style=";text-align:right;direction:rtl"> איבר התורם יוסר הפתרון HTK והשתלות מתחיל osteosynthesis של עצם הירך באמצעות מחט 18-מד (אורך: 1.5-2.0 ס&quot;מ) כמו מוט intramedullary. </li><li style=";text-align:right;direction:rtl"> קבוצות שרירים הגחון הם מקורב באופן הדוק עם Vicryl 4-0 או תפרים Prolene. </li><li style=";text-align:right;direction:rtl"> Anastomoses כלי הדם מבוצעות על ידי משיכת וריד התורם מעל עורק לווריד הנמענים ומורכב השרוול. ליגטורה ההיקפי 8-0 משי ממוקמת שוב כדי לאבטח את השקה. </li><li style=";text-align:right;direction:rtl"> מהדק ורידי הוא שוחרר לאחר מכן תפס העורקים, ו reperfusion הוא מדמיין על ידי החזרת צבע האיבר הדיסטלי רגל ו, כמו גם תזרים pulsatile דרך העורק. </li><li style=";text-align:right;direction:rtl"> הקבוצות הנותרות שריר הגב הם מחדש עם מקורב Vicryl 4-0 או תפרים Prolene. </li><li style=";text-align:right;direction:rtl"> העור הוא סגור עם vicryl 4-0 או תפרים prolene. </li><li style=";text-align:right;direction:rtl"> החולדה הנמען מושם על כרית חימום פיקוח הדוק עד להחלמה. </li><li style=";text-align:right;direction:rtl"> בעלי חיים הנמען לקבל אנטיביוטיקה מניעתית (Cefazolin, 20 מ&quot;ג / יום, IM) ו Buprenex (0.1 מ&quot;ג / ק&quot;ג, SC) במשך שלושה ימים posttransplant. </li></ol>

<ol>
	<li>Shapiro, R. I., Cerra, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+reimplantation+and+transplantation+of+a+complex+organ:+the+rat+hind+limb.">A model for reimplantation and transplantation of a complex organ: the rat hind limb.</a> J Surg Res. 24 (6), 501-506 (1978).</li><li>Petruzzo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+international+registry+on+hand+and+composite+tissue+transplantation.">The international registry on hand and composite tissue transplantation.</a> Transplantation. 86 (4), 487-492 (2008).</li><li>Schneeberger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+forearm+transplantation:+outcome+at+3+years.">First forearm transplantation: outcome at 3 years.</a> Am J Transplant. 7 (7), 1753-1762 (2007).</li></ol>

אין ניגודי אינטרסים הכריז.

עשרות שנים של מחקר ניסיוני יש שקדמה ואפשרה יד השתלה מוצלחת האדם בתקופה האחרונה. בשנת 1978, שפירא ואח&#39;. פיתחו השתלת orthotopic האחוריות איבר מודל בחולדות, מתאים גם ללמוד היבטים פתולוגיים, פיזיולוגיים, ואת מערכת החיסון שונים, כמו גם התוצאה התפקודית של שתלי רקמה מרוכבים. אנחנ?...

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orthotopic hind-limb transplantation in rats

Allotransplantation רקמות Composite (CTA) עכשיו מהווה אופציה טיפולית תקף לאחר אובדן של האמה ביד, או ספרות הפכה לישות טיפולית חדשנית של ניתוח שיקומי. עם זאת, לטווח ארוך במינון גבוה סמים רב טיפול לדיכוי המערכת החיסונית נדרשת כדי להבטיח הישרדות השתל, הנושאת את הסיכון של תופעות לוואי חמורות אשר קולר יישום רחב יותר. התקדמות נוספת בתחום זה תלויה הבנה טובה יותר של בסיסי אימונולוגיה reperfusion איסכמיה פגיעה ברקמות שתלי מרוכבים. נכון להיום, השתלת איבר orthotopic האחוריות של חולדות כבר מכרסם את המודל המועדף שחזור השתלה (RT), לעומת זאת, הוא הליך מאוד תובעני שדורש מיומנויות microsurgical יוצאת דופן וחיבור מחדש של כלי הדם, העצמות, השרירים והעצבים. יש לנו הציג את הטכניקה וסקולרית השקה השרוול מודל זה, מתן גישה מהירה ואמינה עכברוש השתלת איבר האחוריות. טכניקה זו מפשטת ומקצרת את הליך כירורגי ומאפשרת למנתחים בעלי ניסיון בסיסי microsurgical בהצלחה לבצע את הפעולה עם הישרדות גבוהים שיעורי סיבוכים נמוך. הטכניקה נראית גם מתאים החיסונית, כמו גם פגיעה reperfusion איסכמיה (IRI) מחקרים.

Watch this Scientific Journal Video about Orthotopic Hind-Limb Transplantation in Rats at JoVE.com

Orthotopic Hind-Limb Transplantation in Rats

כאן אנו מתארים את העכברוש orthotopic איבר האחוריות, הליך ההשתלה, אשר נראה כי תקן הזהב<em&gt; In vivo</em&gt; מודל למחקר רקמות מרוכבים allotransplantation.

השתלת orthotopic לימב הינד-בחולדות

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

orthotopic-hind-limb-transplantation-in-rats

Research

JoVE Journal

Immunology and Infection

11.6K Views.  Innsbruck Medical University. כאן אנו מתארים את העכברוש orthotopic איבר האחוריות, הליך ההשתלה, אשר נראה כי תקן הזהב In vivo מודל למחקר רקמות מרוכבים allotransplantation.

Video: Orthotopic Hind-Limb Transplantation in Rats

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

בשיטת ההדמיה vivo להבחין דלקת אקוטית וכרונית

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

כרוני לטווח ארוך<em&gt; Pseudomonas aeruginosa</em&gt; זיהום דרכי הנשימה בעכברים

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Thymectomy מלא בחולדות מבוגרות עם לא פולשני endotracheal אינטובציה

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Murine הינד לימב ארוך עצם Dissection ובידוד מח העצם

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

השתלת עור בעובי מלא Murine

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

במבחנה In Vivo למזהמים סביבתיים T, B והתאים מיאלואידית פעילות מדכא ומענים ההורמונאלית מן המקבלים

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

מודל אימונוולוגי עבור הלב הטרוולפית ושריר הלב השתלת תא בחולדות

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

אופטימיזציה של טכניקת השרוול עבור השתלת לב מוריין

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

הליכים השתלת מח עצם בעכברים ללמוד Hematopoiesis שיבוט

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

טיהור והתרחבות של עכבר Invariant טבעי רוצח תאי T עבור במבחנה ו in vivo מחקרים

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...