Donor Betrieb und Hind-Limb Ernte <ol><li> Für das operative Vorgehen eine sterile Reihe von mikrochirurgischen Instrumenten eingesetzt wird. Der Chirurg müssen geeignete Kleidung einschließlich sterile Handschuhe, die vor Beginn des chirurgischen Eingriffs geändert werden müssen. </li><li> Die Ratte ist betäubt mit einer intraperitonealen Injektion von Pentobarbital (50mg/kg IP) und legte in Rückenlage auf einem beheizten Operationsfeld, die eine stabile Tier Körpertemperatur erleichtert über den gesamten chirurgischen Eingriffs. </li><li> Vor Hautschnitt den Betrieb gestellt wird dreimal mit je sowohl saniert, ein Desinfektionsmittel und Alkohol. Um zu verhindern, die Tiere Hornhaut vor dem Austrocknen, die Augen sind mit einer Augensalbe (Vidisic) vor der Operation geschmiert. </li><li> Der Hautschnitt wird über der rechten Leiste etwa auf dem Niveau des Leistenbandes gemacht. Die Femoralgefäße sind frei präpariert mit bipolarer Kauter und stumpf; tiefe Äste werden mit 8-0 Seide ligiert. </li><li> 300μl (50 IU) von Heparin erfolgt über den Penis Vene für Antikoagulation injiziert. </li><li> Femoralgefäße werden auf der Ebene des Leistenbandes ligiert und schneiden distal der Ligatur. </li><li> Das Hinterbein ist mit gerötetem eiskalter (4 ° C) HTK-Lösung (5 ml) über die Oberschenkelarterie über 5 Minuten. </li><li> Alle Muskelgruppen werden stark mit der Schere geteilt und Osteotomie wird mit einer rotierenden elektrischen Säge auf der Ebene des distalen Drittel des Femurs durchgeführt. </li><li> Die geernteten Glied ist dann in 20 mL eiskaltem HTK-Lösung für die Erhaltung und Kühllager bis zur Transplantation untergetaucht. </li></ol> Empfänger Betrieb und Transplantation <ol><li> Die Ratte ist betäubt mit einer intraperitonealen Injektion von Pentobarbital und legte sich rücklings auf den OP-Feld. </li><li> Der Hautschnitt wird über der rechten Leiste etwa auf dem Niveau des Leistenbandes gemacht. Die Femoralgefäße sind frei präpariert mit bipolarer Kauter und stumpf. </li><li> Femoralgefäße als distal wie möglich unterbunden, aber proximal der epigastrische Gefäß Start. Vascular Klemmen sind auf der Ebene des Leistenbandes auf Arterie und Vene gelegt. </li><li> 0,010 mm, Stulpe Länge:;: und Vene (innen Manschettendurchmesser Manschette-Wandstärke 1,151 mm A Polyethylen Manschette über der Arterie (: 0,912 mm, Stulpe-Wandstärke: 0,010 mm, Stulpe Länge 2,0 mm innen Manschettendurchmesser) gezogen : 2,0 mm). Der Rand der Gefäßlumen wird dann mit einem Gefäßdilatator gezogen und der Arteria und Vena femoralis sind über den ganzen Körper der Manschette (ca. 2mm) umgestülpt und mit einer umlaufenden 8-0 Seidenfaden. </li><li> Um Platz zu schaffen für den Spender des Körpers der Empfänger des Körpers ist mit scharfer Präparation von Muskelgruppen entfernt und Osteotomie wird in der Mitte Oberschenkel durchgeführt. </li><li> Der Spender des Körpers wird von der HTK-Lösung entfernt und Transplantation beginnt mit Osteosynthese des Femurs mit einer 18-Gauge-Nadel (Länge: 1,5-2,0 cm) als intramedulläre Stange. </li><li> Ventral Muskelgruppen sind eng mit 4-0 Vicryl oder Prolene Einzelknopfnähten approximiert. </li><li> Gefäßanastomosen werden, indem der Spender Vene und Arterie über die Empfänger Vene und Manschette komplexen durchgeführt. Eine umlaufende 8-0 Seidenfaden wird wieder aufgestellt, um die Anastomose zu sichern. </li><li> Die venöse Klemme gelöst, gefolgt von der arteriellen Klemme, und Reperfusion wird visualisiert durch Rücksendung der Farbe auf die distalen Gliedmaßen und Fuß sowie pulsatilen Fluss durch die Arterie. </li><li> Die restlichen dorsalen Muskelgruppen werden mit 4-0 Vicryl oder Prolene Einzelknopfnähten wieder angenähert. </li><li> Die Haut ist mit 4-0 Vicryl oder Prolene Einzelknopfnähten verschlossen. </li><li> Der Empfänger Ratte befindet sich auf einem Heizkissen gelegt und eng bis zur Genesung überwacht. </li><li> Empfänger Tiere prophylaktisch Antibiotika (Cefazolin, 20 mg / Tag, IM) und Buprenex (0,1 mg / kg, SC) für drei Transplantation Tage. </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>

Jahrzehntelange experimentelle Forschung vorausgegangen und ermöglichte erfolgreiche menschliche Hand-Transplantation in der jüngsten Vergangenheit. Im Jahr 1978, Shapiro et al. entwickelte eine orthotope Hinterbein Transplantation Modell in Ratten, gut geeignet, um verschiedene pathologische, physiologische und immunologische Aspekte sowie funktionelle Ergebnis von Composite Gewebetransplantate studieren. Wir haben den schwierigsten Teil des Verfahrens, nämlich die Gefäßanastomose geändert durch die Einführung e...

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orthotopic hind-limb transplantation in rats

Composite-Gewebe Allotransplantation (CTA) stellt nun eine gültige therapeutische Option nach dem Verlust einer Hand, Unterarm oder Ziffern und hat sich zu einem neuen therapeutischen Einheit in der rekonstruktiven Chirurgie. Allerdings ist langfristig hochdosierten Multi-Drug-immunsuppressive Therapie erforderlich, um Transplantat Überleben zu sichern, wobei das Risiko von schweren Nebenwirkungen, die eine breitere Anwendung Halfter. Weitere Progression in diesem Bereich kann auf ein besseres Verständnis der Grundlagen der Immunologie und Ischämie Reperfusionsschaden in Composite Gewebetransplantate ab. Bis heute orthotopen Hinterbein Transplantation in Ratten wurde die bevorzugte Nagermodell für die rekonstruktive Transplantation (RT) wurde, ist es jedoch ein sehr anspruchsvolles Verfahren, das außergewöhnliche mikrochirurgische Fähigkeiten erfordert für Wiederbefestigung der Gefäße, Knochen, Muskeln und Nerven. Wir haben die vaskuläre Manschette Anastomosentechnik diesem Modell eingeführt, die eine schnelle und zuverlässige Methode, um Hinterbein Transplantation Ratte. Diese Technik vereinfacht und verkürzt den chirurgischen Eingriff und können Chirurgen mit grundlegenden mikrochirurgische Erfahrung, um erfolgreich die Operation mit hoher Überlebens-und niedrigen Komplikationsraten. Die Technik scheint auch für immunologische sowie Ischämie Reperfusionsschaden (IRI) Studien geeignet sein.

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

Orthotopic Hind-Limb Transplantation in Rats

Hier beschreiben wir die orthotopen Ratten-Hinterteil des Körpers Transplantation Verfahren, das an den Goldstandard zu sein scheint<em&gt; In vivo</em&gt; Modell für Composite Tissue Allotransplantation Forschung.

Orthotopen Hind-Limb Transplantation in Ratten

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 Aufrufe.  Innsbruck Medical University. Hier beschreiben wir die orthotopen Ratten-Hinterteil des Körpers Transplantation Verfahren, das an den Goldstandard zu sein scheint In vivo Modell für Composite Tissue Allotransplantation Forschung.

Serie: Orthotopic Hind-Limb Transplantation in Rats

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Diagnose von Ekto-und Endoparasiten in Laborratten und Labormäusen

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Forschung

Immunologie und Infektion

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.

In-vivo-Imaging-Methode zu unterscheiden, akuten und chronischen Entzündungen

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.

Langzeitchronik<em&gt; Pseudomonas aeruginosa</em&gt; Airway-Infektion bei Mäusen

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.

Komplette Thymektomie bei erwachsenen Ratten mit Nicht-invasive Intubation

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 Hind Gliedmaßen langen Knochen Dissection und Knochenmark-Isolation

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

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.

In Vitro und In Vivo Beurteilung der T, B und myeloische Zellen Suppressive Aktivität und humorale Reaktionen von Transplantationspatienten

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.

Ein immunologisches Modell für heterotopische Herz- und Herzmuskelzelltransplantation bei Ratten

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.

Optimierung der Manschettentechnik für DieMurine Heart Transplantation

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.

Knochenmarktransplantationsverfahren bei Mäusen zur Untersuchung der klonalen Hämatopoese

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.

Aufreinigung und Expansion von mausinvarianten natürlichen Killer-T-Zellen für in vitro und in vivo Studien

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