Questo lavoro è stato sostenuto in parte dal NIH concedere AI069358 e la Fondazione di Ricerca BloodCenter.

<ol><li> Prima di avviare il processo, i seguenti elementi devono essere raccolte e preparate per: <ul><li> Ghiaccio </li><li> Siringa da 5 ml con ago 27g </li><li> Siringa da 5 ml con ago 25g </li><li> Blocco di polistirolo e perni per il montaggio del mouse </li><li> Vassoio sul quale il blocco di montaggio può essere posizionato </li><li> Forbici e pinze </li><li> Raccolta tubi </li><li> Etanolo al 70% </li><li> PBS con il 3% di siero fetale bovino (FCS) (Pre refrigerati e tenuta in ghiaccio) </li></ul></li><li> Euthanize il mouse, spruzzare con il 70% di etanolo e montarlo sul blocco di polistirolo sulla sua schiena. </li><li> Utilizzando un paio di forbici e pinze tagliare la pelle esterna del peritoneo e delicatamente tirare indietro per esporre la pelle interna che riveste la cavità peritoneale. </li><li> Iniettare 5 ml di PBS freddo ghiaccio (con il 3% FCS) nella cavità peritoneale con un ago 27g. Spingere l&#39;ago lentamente nel peritoneo facendo attenzione a non forare qualsiasi organi. </li><li> Dopo l&#39;iniezione, massaggiare delicatamente il peritoneo per rimuovere tutte le cellule attaccato nella soluzione PBS. </li><li> Inserire un ago da 25 g, smusso fino, attaccato ad una siringa da 5 ml nel peritoneo e raccogliere il liquido mentre si muove la punta dell&#39;ago delicatamente per evitare di intasare dal tessuto adiposo o di altri organi. Raccogliere quanto più possibile fluido e depositare la sospensione cellulare raccolti in provette tenuta in ghiaccio dopo aver rimosso l&#39;ago dalla siringa. Opzionale: Ripetere il punto 4-6 </li><li> Fare un&#39;incisione nella pelle interna del peritoneo e tenendo la pelle con una pinza usare una pipetta Pasteur di plastica per raccogliere il liquido rimanente dalla cavità. </li><li> Se al punto 6 o 7 contaminazione del sangue visibile viene rilevato poi il campione contaminato deve essere eliminato. </li><li> Gira la sospensione cellulare raccolti a 1500 RPM per 8 minuti, scartare il sopranatante e risospendere le cellule nel supporto desiderato o PBS per il conteggio. </li></ol> Protocollo alternativo di ottenere Thioglycollate suscitato macrofagi: Questo metodo è utilizzato per ottenere una maggiore resa dei macrofagi. <ol><li> Iniettare 5 ml di 3% (w / v) Brewer Thioglycollate media (7) nella cavità peritoneale di ogni topo. </li><li> Attendere 3-5 giorni, e passare al punto 1 di cui sopra per la raccolta delle cellule. </li></ol> Rispetto all&#39;approccio nonelicited, circa 10 volte più macrofagi possono essere raccolti. L&#39;unica preoccupazione è che i macrofagi ottenuti da questa procedura si differenziano per alcune delle loro proprietà fisiologiche. Rappresentante dei risultati: Da un mouse non manipolata, 5-10 milioni di cellule cavità peritoneale può essere ottenuto con un protocollo di buon isolamento. Fra tutte le cellule vive, il 50-60% sono cellule B, macrofagi ~ 30% e 5-10% sono cellule T (Fig. 1). <img src="/files/ftp_upload/1488/1488fig1.jpg" alt="Figura 1" /> Figura 1. Fenotipo delle cellule isolate dalla cavità peritoneale. Isolamento seguito delle cellule cavità peritoneale sono stati colorati con anti-topo TCRβ-FITC, B220-PE-Texas rosso-blu del Pacifico CD11b, CD23-PE-Cy7 e CD5-APC. Trame rappresentante citometria di flusso mostrano percentuali di cellule B e T (a), macrofagi (b), B1 e B2 cellule (c) e le cellule B1a e B1b (d).

<ol><li>Zhang, X., Goncalves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolation+and+characterization+of+murine+macrophages%5BTitle%5D)+AND+%22Current+Protocols+in+Immunology%22%5BJournal%5D)">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/pubmed?term=((A+critical+role+of+natural+immunoglobulin+M+in+immediate+defense+against+systemic+bacterial+infection%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">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/pubmed?term=((Blood+clearance+by+anti-phosphocholine+antibodies+as+a+mechanism+of+protection+in+experimental+pneumococcal+bacteremia%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">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/pubmed?term=((B1+B+lymphocytes+play+a+critical+role+in+host+protection+against+lymphatic+filarial+parasites%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">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/pubmed?term=((Normal+mouse+peritoneum+contains+a+large+population+of+Ly-1%2B+%28CD5%29+B+cells+that+recognize+phosphatidyl+choline%3A+relationship+to+cells+that+secrete+hemolytic+antibody+specific+for+autologous+erythrocytes%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">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/pubmed?term=((Ly-1+B+%28B-1%29+cells+are+the+main+source+of+B+cell+derived+interleukin+10%5BTitle%5D)+AND+%22Eur.+J.+Immunol%22%5BJournal%5D)">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/pubmed?term=((Macrophage+activation+to+kill+Leishmania+tropica%3A+Defective+intracellular+killing+of+amastigotes+by+macrophages+elicited+with+sterile+inflammatory+agents%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+the+factor+in+L-cell+conditioned+medium+capable+of+stimulating+colony+formation+by+mouse+marrow+cells+in+culture%5BTitle%5D)+AND+%22J.+Cell+Physiol%22%5BJournal%5D)">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/pubmed?term=((The+macrophage+colony-stimulating+factor%2C+CSF-1%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">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/pubmed?term=((The+role+of+B-1+and+B-2+cells+in+immune+protection+from+influenza+virus+infection%5BTitle%5D)+AND+%22Curr.+Top.+Microbiol.+Immunol%22%5BJournal%5D)">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/pubmed?term=((B-1+and+B-2+cell-derived+immunoglobulin+M+antibodies+are+nonredundant+components+of+the+protective+response+to+influenza+virus+infection%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">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/pubmed?term=((Origins+and+functions+of+B-1+cells+with+notes+on+the+role+of+CD5%5BTitle%5D)+AND+%22Annu.+Rev.+Immunol%22%5BJournal%5D)">Origins and functions of B-1 cells with notes on the role of CD5.</a> Annu. Rev. Immunol. 20, 253-300 (2002).</li>
</ol>

Isolamento di cellule cavità peritoneale è una tecnica importante per lo studio delle diverse cellule del sistema immunitario, principalmente i macrofagi e specifici sottoinsiemi delle cellule B. Anche se questo è un processo semplice, ci sono alcuni passaggi critici coinvolti. Presenza di contaminanti nel sangue raccolto il campione deve essere evitato per ottenere una pura cavità peritoneale popolazione di cellule. Eutanasia per dislocazione cervicale dovrebbe essere fatto con attenzione per evitare la contaminazione del sangue nella cavità peritoneale. In alternativa CO 2 può essere utilizzato. Inoltre, durante la cura corretta procedura da adottare per evitare la puntura della vescica o degli altri organi nella cavità peritoneale. Recuperare la maggior parte del liquido iniettato è importante anche per il rendimento della cella. Questa procedura è ampiamente utilizzato per studiare la biologia dei macrofagi dal numero moderato di residenti, i macrofagi non stimolate possono essere facilmente ottenuto dalla cavità peritoneale, in contrasto con il laborioso compito di differenziazione delle cellule progenitrici mieloidi in macrofagi maturi in vitro, utilizzando fattore stimolante le colonie di macrofagi (8 , 9). La resa dei macrofagi dalla cavità peritoneale può essere migliorata utilizzando il metodo di elicitazione Thioglycollate, anche se l&#39;uso Thioglycollate potrebbero alterare le proprietà fisiologiche dei macrofagi (7). Cellule B sono una parte importante del nostro sistema immunitario giocare un ruolo cruciale sia l&#39;immunità innata e adattativa. Tra le diverse sottopopolazioni di linfociti B, cellule B1 costituiscono un particolare sottogruppo di cellule B coinvolte nell&#39;immunità innata, autoimmunità e regolazione del sistema immunitario. Celle B1 sono principalmente situati nella cavità peritoneale e rifornimento di auto. Si tratta di uno dei produttori primari di IgM naturali che fornisce la prima linea di protezione contro un certo numero di virus e batteri (10-12). Celle B1 sono anche una fonte importante di IL-10 (6), che è una citochina importante coinvolte nella regolazione del sistema immunitario. Anche se diversi studi sono stati perseguiti con le cellule B1, c&#39;è ancora spazio per un&#39;ulteriore valutazione delle loro funzioni contrastanti, specialmente il loro ruolo regolatore. Cavità peritoneale di cellule metodo isolamento offre l&#39;opportunità unica di studiare le funzioni delle cellule B1.

<table><tbody><tr><td>Thioglycollate Medium, Brewer Modified</td><td>BD Biosciences</td><td>211716</td><td></td></tr></tbody></table>

isolation of mouse peritoneal cavity cells

La cavità peritoneale è una cavità legata alla membrana e piene di liquido addominale di mammiferi, che contiene il fegato, nella milza, la maggior parte del tratto gastro-intestinale e altre viscere. Si porti un certo numero di cellule immunitarie tra cui i macrofagi, le cellule B e le cellule T. La presenza di un elevato numero di macrofagi ingenuo nella cavità peritoneale rende un luogo preferito per la raccolta dei macrofagi residenti ingenuo dei tessuti (1). La cavità peritoneale è importante anche per lo studio delle cellule B a causa della presenza di un unico cavità peritoneale residenti sottoinsieme B cellule conosciute come cellule B1, oltre alle cellule B2 convenzionali. Celle B1 sono suddivise in celle B1a e B1b, che si distingue per l&#39;espressione di superficie di CD11b e CD5. Celle B1 sono una fonte importante di IgM fisiche che forniscono protezione precoce da una varietà di agenti patogeni (2-4). Queste cellule sono autoreattivi in ​​natura (5), ma come vengono controllati per evitare che l&#39;autoimmunità non è ancora compresa del tutto. Al contrario, CD5<sup&gt; +</sup&gt; B1a cellule possiedono alcune proprietà normativo in virtù della loro capacità di IL-10 produce (6). Pertanto, le cellule B1 cavità peritoneale sono una popolazione di cellule interessante da studiare a causa della loro funzione diverse e molte domande senza indirizzo associati al loro sviluppo e regolamentazione. L&#39;isolamento di cavità peritoneale cellule immunitarie residenti è difficile a causa della mancanza di una struttura definita all&#39;interno della cavità peritoneale. Il nostro protocollo si descrive una procedura per ottenere vitali cellule immunitarie dalla cavità peritoneale di topi, che poi può essere utilizzato per l&#39;analisi fenotipica mediante citometria di flusso e di diversi saggi biochimici e immunologici.

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

Isolation of Mouse Peritoneal Cavity Cells

La cavità peritoneale nei mammiferi contiene diverse popolazioni di cellule immunitarie cruciali per risposte immunitarie innate. Un metodo efficiente isolamento è necessario per le analisi biochimiche e funzionali di queste cellule. Qui forniamo un metodo completo per l&#39;isolamento di cellule cavità peritoneale nel topo.

Isolamento di cellule della cavità peritoneale mouse

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

143.3K Views.  Blood Research Institute. La cavità peritoneale nei mammiferi contiene diverse popolazioni di cellule immunitarie cruciali per risposte immunitarie innate. Un metodo efficiente isolamento è necessario per le analisi biochimiche e funzionali di queste cellule. Qui forniamo un metodo completo per l'isolamento di cellule cavità peritoneale nel topo.

Video: Isolation of Mouse Peritoneal Cavity Cells

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.

Isolamento di cellule polmonari mouse dendritiche

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 ...

Ricerca

Immunologia e infezioni

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Isolare e Immunostaining linfociti e le cellule dendritiche da placche di Peyer murino di

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

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.

Isolamento di murini peritoneale macrofagi di effettuare analisi di espressione genica Al Toll-like recettori stimolazione

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

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

Isolamento dei mastociti peritoneo-derivati e loro caratterizzazione funzionale con Ca2 +-imaging e degranulazione saggi

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Isolamento e coltura di macrofagi derivati dal midollo osseo da topi

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

<meta charset="utf8"></head><body>Preparazione di vettori lentivirali per la modulazione genica in vivo

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

<meta charset="utf8"></head><body>Autore in primo piano: Esplorazione dell'immunometabolismo dei macrofagi attraverso la manipolazione genica mediata da vettori lentivirali

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

Preparazione di vettori lentivirali per un'efficiente modulazione di geni e microRNA di macrofagi residenti nei tessuti della cavità peritoneale in vivo nei topi

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

Isolamento del mesenchima intestinale murino con conseguente alta resa di telociti

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

Medicina

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

A cellula intera elettrofisiologia di cellule enterocromaffini murino coltura primaria

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Isolamento di cellule della cavità peritoneale mouse

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