Ringraziamo Aaron Rae (Emory University Dipartimento di Pediatria e Healthcare bambini di Core flusso Atlanta) per l&#39;ordinamento delle cellule. Questo lavoro è stato supportato da sovvenzioni NIH AA01787001, un Career Development Award dal morbo di Crohn e Colite Foundation of America, e della ricerca uno Emory-Egleston Children Center di sementi sovvenzione TLD

1. Dissezione e la dissociazione di cellule epiteliali intestinali Preparazione dei reagenti e attrezzature: <ul><li> Caldo Ca 2 + / Mg 2 +-libera PBS (CMF PBS) a temperatura ambiente. </li><li> Caldo Ca 2 + / Mg 2 +-libere HBSS con 5% FBS (CMF HBSS / FBS) e 2 mM EDTA a temperatura ambiente. </li><li> Warm Agitatore orbitale a 37 ° C. </li></ul> Nota: Procedura 1,1 a 1,7 devono essere eseguite più rapidamente possibile per minimizzare l&#39;entità della morte cellulare e per ottenere la massima resa cella. <ol><li> Euthanize topi in una camera di CO 2 e l&#39;etanolo al 70% a spruzzo sul addome e torace. </li><li> Effettuare una piccola incisione orizzontale al centro dell&#39;addome con una forbice e staccare la pelle per esporre il peritoneo. </li><li> Procedere per separare lo stomaco dalla parte superiore dell&#39;intestino tenue tagliando alla sfintere pilorico. Tease via il mesentere con una pinza e tagliare di nuovo al&#39;ileo-cecale valvola per liberare l&#39;intero intestino piccolo crasso. Fare un taglio al margine anale e di nuovo prendere in giro oltre il mesentere fino a quando il grosso intestino è gratuito. </li><li> Tagliare i due punti longitudinalmente con le forbici e lavare i contenuti fecali e muco dal lume intestinale in CMF PBS a temperatura ambiente. </li><li> Usare le forbici e pinze a sezionare macroscopicamente le placche di Peyer, lungo la superficie anti-mesenterico dell&#39;intestino tenue e tagliare aperta longitudinalmente l&#39;intestino tenue. </li><li> Lavare il lume dell&#39;intestino tenue di contenuti fecali e muco nelle CMF PBS a temperatura ambiente. </li><li> A parte tagliare il piccolo intestino / grandi dimensioni in pezzi di circa 1,5 cm e mettetela in distinte provette da 50 ml coniche contenenti 30 ml di pre-riscaldato HBSS CMF / FBS e 2 mM EDTA. Non aggiungere più di 1 intestino per tubo da 50 ml. </li><li> Posizionare orizzontalmente ogni tubo da 50 ml conica in un agitatore orbitale ed agitare per a 250 rpm per 20 min a 37 ° C. &lt;/ Li&gt; <li> Posizionare un unico filtro di rete metallica su un secchio dei rifiuti e versare il contenuto di ciascuna provetta 50 ml conica attraverso il recupero dei pezzi di 1,5 cm di intestino e metterli in diverse provette da 50 ml coniche contenenti 30 ml di pre-riscaldati CHBSS / FB con 2 mM EDTA. </li><li> Ripetere 1,8. </li></ol> 2. Tissue Digestione e isolamento delle cellule intestinali Preparazioni a base di reagenti e attrezzature: <ul><li> Collagenasi soluzione: 1,5 mg / ml Tipo VIII Collagenasi disciolto in preriscaldata HBSS CMF / FBS con 40 pg / ml di DNasi I. </li><li> Pre-riscaldata HBSS CMF / FBS e 2 mM EDTA. </li><li> Pre-riscaldata agitatore orbitale a 37 ° C. </li><li> Ice-freddo HBSS CMF / FBS. </li></ul><ol><li> Dopo il secondo turno di scuotere, versare il contenuto di ciascuna provetta 50 ml conica attraverso il filtro e trasferire pezzi di 1,5 cm di intestino ad una plastica piccola pesa barca dopo tamponando distanza supporti in eccesso con un tovagliolo di carta. </li><li> Rapidamente tritare il 1.5 Pezzi cm di intestino usando forbici direttamente sulla barca peso e aggiungere intestino macinata a 20 ml di soluzione di collagenasi. Posizionare orizzontalmente ogni tubo da 50 ml conica in un agitatore orbitale e digest a 200 rpm per 10-20 min a 37 ° C. Si prega di consultare la discussione sotto per quanto riguarda l&#39;ottimizzazione. </li><li> Brevemente vortice di assicurare la dissociazione completa di qualsiasi restante tessuto intestinale e filtrare attraverso un colino 100 micron cella direttamente in una provetta da 50 ml. </li><li> Top off ogni provetta conica da 50 ml con HBSS CMF / FBS e centrifugare a 1500 rpm per 5 min a 4 ° C. Se un solido pellet non si osserva per i campioni del colon dopo la centrifugazione, i campioni devono essere centrifugati di nuovo per 3,5 min. Ripetere questa fase di lavaggio, una volta di più. </li><li> Eliminare il sopranatante e risospendere il pellet cellulare in ghiacciato HBSS CMF / FBS e campioni posto sul ghiaccio. </li><li> Procedere alla Sezione 3 per l&#39;acquisizione FACS / analisi o la sezione 4 per magneto-tallone di arricchimento per la cella sorti ad alta velocitàng. </li></ol> 3. Colorazione anticorpo per Multi-Color mediante citometria a flusso delle cellule dendritiche e macrofagi Preparazioni a base di reagenti e attrezzature: <ul><li> Ice-freddo CMF PBS. </li><li> Tampone ghiacciato colorazione (CMF PBS + 5% FBS). </li><li> Preparare cellula morta macchia in ghiacciato CMF PBS alla diluizione 1:1000 utilizzando LIVE / DEAD fissabile Kit Dead Cell Aqua Stain. </li><li> Preparare il cocktail di anticorpi colorazione aggiungendo le seguenti fluorescenza marcati anticorpi monoclonali (mAb) al tampone ghiacciato colorazione: PerCP CD45-, CD103-PE, CD11c-APC, MHC-II (IA b)-Alexa Fluor 700, CD11b eFluor-450, F4/80-PE-Cy7. </li></ul><ol><li> Trasferire le cellule in un polistirene da 5 ml a fondo rotondo (FACS) tubo. </li><li> Lavare le cellule due volte in ghiacciato CMF PBS. </li><li> Incubare campioni con cellule morte colorare per 15 min in ghiaccio al buio. </li><li> Lavare le cellule due volte in ghiacciato CMF PBS. </li><li> Celle a blocchi con 2.4G2 anti-FcγRIII/II in ghiacciato colorazionetampone per 10 min in ghiaccio. </li><li> Lavare le cellule in tampone ghiacciato colorazione. </li><li> Incubare campioni con cocktail colorazione anticorpale per 20 min in ghiaccio al buio. </li><li> Lavare le cellule con tampone ghiacciato colorazione due volte campioni e risospendere in 400 microlitri di tampone ghiacciato colorazione e passare attraverso cap 40 micron filtro sui tubi FACS. </li><li> Acquisire campioni su LSR II citometro (BD), come definiti dalla strategia di gating nella Sezione 5 e nella Figura 1. </li></ol> 4. Arricchimento della DC e dei macrofagi da parte dell&#39;intestino Preparazioni a base di reagenti e attrezzature: <ul><li> Tampone ghiacciato colorazione (CMF PBS + 5% FBS). </li></ul><ol><li> Incubare singola sospensione cellulare ottenuta dal punto 2.5 con CD11b e CD11c perle MACS secondo le istruzioni del produttore. </li><li> Lavare le cellule con tampone ghiacciato colorazione seguita da centrifugazione. </li><li> Gettare il surnatante e risospendere il pellet di cellule in 1 ml di tampone ghiacciato colorazionee passano attraverso un filtro 100 pm cella seguito da un filtro cella 40 um. </li><li> Arricchire per magnetici bead-cellule collegate da una selezione positiva utilizzando MACS colonna magnetica LS. </li><li> Ripetere il passaggio 4.2 e scartare il surnatante. </li><li> Incubare le cellule con anticorpi monoclonali marcatori di superficie come descritto al punto 3.7. </li><li> Lavare magnetici tallone arricchiti di cellule due volte con tampone ghiacciato colorazione. Risospendere pellet cellulari in 500 microlitri tampone ghiacciato colorazione senza azide di sodio, e passare attraverso il filtro 40 micron cellule in una provetta FACS. </li><li> Procedere alla FACS-ordinamento sulla cella BD ARIA Sorter II per ordinare DC intestinale e / o sottoinsiemi dei macrofagi di interesse. </li></ol> 5. Strategia di gating per LP APC Nota: Si noti che non marcate cellule intestinali può essere utilizzato come controllo negativo per assistere nel posizionamento corretto delle porte per separare popolazioni positive e negative. <ol><li> Come mostrato in figura1, creare un grafico punto e escludere le cellule che sono positivi per la cella morti macchia (Fig. 1A) seguita dalla esclusione di eventi doppietto (Fig. 1B e C). Poi, porta sulle cellule di interesse in funzione di trasmettere e disperdere lato fare in modo di escludere i detriti (Fig. 1D). </li><li> Creare un altro dot plot e porta avanti CD45 + b + e IA, che caratterizza le cellule fenotipicamente APC (Fig. 1E). </li><li> Su un diagramma dot separato, per analizzare l&#39;espressione CD11c CD11b e per distinguere DC specifici sottoinsiemi e macrofagi (R1, R2, e R3;. Figura 1F). Cellule di R1 sono CD11c + CD11b spenti / - cellule. La regione, R2, è delineato in modo tale che queste cellule hanno livelli simili di espressione superficie CD11c come in cellule di R1, ma anche cellule di R2 esprimono CD11b. Successivamente, la regione R3 è designato per le celle che sono + CD11b e opaco / CD11c -. </li><li> Unalyze regioni R1, R2, R3 e ulteriore espressione per F4/80 e CD103 differenziare macrofagi e popolazioni CC, rispettivamente. Il CD11c + CD11b opaco / - cellule R1 esprimono alti livelli di integrina αE, CD103, e bassi livelli di F4/80 (Fig. 1G). CD11b + + CD11c cellule di R2 sono composti da entrambi i DC e dei macrofagi in base alla loro espressione dicotomica del CD103 e F4/80 (Fig. 1 H). Infine, CD11b + CD11c opache / - cellule di macrofagi R3 costituiscono in base al profilo di fenotipica F4/80 CD103 + e - (Fig. 1I) 16. </li></ol> 6. Risultati rappresentativi <img alt="Figura 1" src="/files/ftp_upload/4040/4040fig1.jpg" /> Figura 1. Strategia di gating per DC intestinali e macrofagi. Cellule morte (A) e farsetti (B e C) sono stati esclusi dall&#39;analisi e poi piccolo intestinalenali cellule erano gated conseguenza di trasmettere e lato scatter (D), e APC sono stati definiti come CD45 + IA b + (E). I macrofagi e DC sono stati identificati mediante l&#39;espressione di CD11b e CD11c (F). CD103 e F4/80 espressione per cellule pre-gated su R1 (G), R2 (H) e R3 (I) popolazioni stato analizzato. <img alt="Figura 2" src="/files/ftp_upload/4040/4040fig2.jpg" /> Figura 2. Quantità di cellule e la colorazione di qualità anticorpi dipende dal tempo di digestione. CD11b e pattern di colorazione CD11c e la resa totale delle cellule di sotto-(A, D), in modo ottimale-(B, E) o sopra-digerito (C, F) tessuto intestinale. Cellule intestinali sono stati isolati da un intestino C57BL / 6 topo piccolo e DC e macrofagi sono stati analizzati mediante FACS sulla LSR BD II. Tensione e compensazione erano state impostate utilizzando solo fluorocromo non colorati e macchiati di splenociti. Le cellule morte (Fig. 1A) e farsetti (Fig. 1B e C) sono stati esclusi dallal&#39;analisi. Le cellule di interesse sono stati poi analizzati secondo di trasmettere e lato scatter (Fig. 1D), seguita da gating sul CD45 + b + IA e cellule (Fig. 1E). Successivamente, CD11b ed espressione CD11c è stata valutata tra il CD45 + b + IA cellule per delineare tre regioni (R1, R2, e R3;. Figura 1F). CD103 e F4/80 espressione nelle tre regioni è stata valutata per distinguere tra DCS e macrofagi, rispettivamente. Il CD11c + CD11b / opaco - cellule R1 espresso alti livelli di integrina αE, CD103, e bassi livelli di F4/80 (Fig. 1G). CD11b + + CD11c cellule R2 erano composti sia DC e dei macrofagi in base alla loro espressione dicotomica del CD103 e F4/80 (Fig. 1 H), mentre CD11b + CD11c spenti / - cellule R3 costituiscono macrofagi in base al profilo fenotipica della F4 / 80 + e CD103 - ( <strong&gt; fig. 1I) 16. I macrofagi all&#39;interno del cancello R2 e macrofagi nel cancello R3 in avanti hanno proprietà simili e dispersione laterali e si distinguono per l&#39;espressione CD11c. La dicotomia funzionale di questi sottoinsiemi rimane completamente sconosciuto. Il rapporto tra la durata di digestione tessutale sulla resa cellulare totale e l&#39;espressione di CD11b e CD11c è illustrato nella figura 2. Tessuto intestinale che è stato digerito per 3 min (sotto-digestione) prodotto a basso numero totale delle cellule (Fig. 2D) e DCS così pochi e macrofagi disponibili per la caratterizzazione (Fig. 2A). La digestione del tessuto per 11 minuti ha prodotto un rendimento affidabile di cellule vive (Fig. 2E) con popolazioni di cellule dendritiche ei macrofagi che esprimono alti livelli di CD11b e CD11c ed erano fenotipicamente distinti (Fig. 2C). Al contrario, la digestione per 50 min (over-digestione) ha comportato una resa simile cella quando compared a digestione ottimizzata (2E Fig. e F), tuttavia, la definizione delle diverse popolazioni cellulari utilizzando CD11b e CD11c divenne più alternativi come l&#39;espressione di diminuita CD11c (Fig. 2C) e il numero di cellule morte aumentata (dati non mostrati).

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<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="Figura 3"/> Figura 3. Fattori importanti per l&#39;ottimizzazione della resa delle cellule e l&#39;espressione dell&#39;antigene di superficie. Resa cellulare e l&#39;espressione dell&#39;antigene di superficie sono direttamente interessati dalla durata della digestione dei tessuti, le caratteristiche specifiche del collagenasi, il grado di macinazione dei tessuti e la presenza o l&#39;assenza di infiammazione, che possano pregiudicare l&#39;integ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> 1X PBS, Ca 2 + - 2 + e Mg-free </td><td></td><td></td><td></td></tr><tr><td> Sale di Hank soluzione equilibrata (HBSS) con rosso fenolo </td><td> Fisher Scientific </td><td> SH3001603 </td><td></td></tr><tr><td> Bicarbonato di sodio </td><td> Sigma </td><td> S6014 </td><td></td></tr><tr><td> HEPES 1M in 0,85% NaCl </td><td> Lonza </td><td> 17-737E </td><td></td></tr><tr><td> Siero bovino fetale (FBS) </td><td> Atlanta biologici </td><td> S11150H </td><td> Inattivato al calore </td></tr><tr><td> 0,5 M EDTA (pH 8,0) </td><td> Cellgro </td><td> 46-034-CI </td><td></td></tr><tr><td> Collagenase di tipo VIII </td><td> Sigma </td><td> C2139 </td><td></td></tr><tr><td> DNasi I </td><td> Roche </td><td> 14785000 </td><td> Soluzione madre: 100mg/ml </td></tr><tr><td> LIVE / DEAD fissabile Aqua Kit Dead Cell Stain per 405 nm di eccitazione </td><td> Invitrogen </td><td> L34957 </td><td> Utilizzare a 1:1000 </td></tr><tr><td> CD45-PerCP mAb (30F11) </td><td> BD </td><td> 557235 </td><td> Utilizzare a 1:100 </td></tr><tr><td> CD103-PE mAb (M290) </td><td> BD </td><td> 557495 </td><td> Utilizzare a 1:100 </td></tr><tr><td> FcyRIII / II mAb (2.4G2) </td><td> BD </td><td> 553141 </td><td> Utilizzare a 1:200 </td></tr><tr><td> CD11c-APC mAb (N418) </td><td> eBioscience </td><td> 17-0114-82 </td><td> Utilizzare a 1:100 </td></tr><tr><td> MHC-II (I-Ab)-Alexa Fluor 700 mAb </td><td> eBioscience </td><td> 56-5321-82 </td><td> Utilizzare a 1:100 </td></tr><tr><td> CD11b-eFluor 450mAb (M1/70) </td><td> eBioscience </td><td> 48-0112-82 </td><td> Utilizzare a 1:200 </td></tr><tr><td> F4/80-PE-Cy7 mAb (BM8) </td><td> eBioscience </td><td> 25-4801-82 </td><td></td></tr><tr><td> CD11b microsfere </td><td> Miltenyi Biotec </td><td> 130-049-601 </td><td></td></tr><tr><td> CD11c microsfere </td><td> Miltenyi Biotec </td><td> 130-052-001 </td><td></td></tr><tr><td> 50 ml provette coniche </td><td> BD Falcon </td><td> 352098 </td><td></td></tr><tr><td> Singola mesh filo </td><td> Chefmate </td><td></td><td></td></tr><tr><td> Piccola pesano barca </td><td> Fisher Scientific </td><td> 08-732-116 </td><td></td></tr><tr><td> Filtro da 100 micron delle cellule </td><td> BD Falcon </td><td> 352360 </td><td></td></tr><tr><td> 40 micron filtro cella </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 5 ml in polistirolo a fondo tondo tubi </td><td> BD Falcon </td><td> 352235 </td><td> Utilizzare a 1:100 </td></tr><tr><td> MaxQ 4450 agitatore orbitale da banco </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> LS MACS colonna </td><td> Miltenyi Biotec </td><td> 130-042-401 </td><td></td></tr><tr><td> LSR II </td><td> BD </td><td></td><td></td></tr><tr><td> FACSAria II </td><td> BD </td><td></td><td></td></tr></tbody></table>

isolation and characterization of dendritic cells and macrophages from the mouse intestine

All&#39;interno l&#39;intestino risiedono popolazioni uniche di cellule immunitarie innate e adattiva che sono coinvolti nella promozione della tolleranza verso la flora commensale e gli antigeni alimentari, mentre in concomitanza rimanendo in bilico per montare le risposte infiammatorie verso invasive 1,2 patogeni. Cellule presentanti l&#39;antigene, in particolare DCS e macrofagi, giocano un ruolo critico nel mantenimento della omeostasi immunitaria intestinale attraverso la loro capacità di percepire e rispondere adeguatamente al microbiota 3-14. Isolamento efficiente di DC intestinali e macrofagi è un passo fondamentale nel caratterizzare il fenotipo e la funzione di queste cellule. Mentre molti metodi efficaci per isolare cellule immunitarie intestinali, compresi DC e macrofagi, sono stati descritti 6,10,15-24, contare su lunghe molte volte digestioni che possono influenzare negativamente l&#39;espressione sulla superficie cellulare antigene, la vitalità cellulare, e / o quantità di cellule. Qui, si dettaglio una metodologia per l&#39;isolamento rapido di un gran numero di viablelettronici, DCS intestinali e macrofagi. Caratterizzazione fenotipica delle DC intestinali e macrofagi viene effettuata direttamente colorazione cellule intestinali isolate con specifici anticorpi monoclonali marcati a fluorescenza per multi-colore analisi citofluorimetrica. Inoltre, DC altamente puro e popolazioni macrofagi sono isolati per studi funzionali utilizzano CD11c e CD11b magnetico attivati ​​perline di separazione delle cellule seguite da selezione cellulare.

Watch this Scientific Journal Video about Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine at JoVE.com

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

Qui, dettaglio una metodologia per l&#39;isolamento rapido delle cellule dendritiche di topo intestinali (DCS) e macrofagi. Caratterizzazione fenotipica di DC intestinali e macrofagi viene eseguita utilizzando multi-color analisi citometrica mentre magnetica arricchimento tallone seguita dalla selezione cellulare viene utilizzato per produrre le popolazioni di elevata purezza per gli studi funzionali.

Isolamento e caratterizzazione di cellule dendritiche ei macrofagi dall&#39;intestino mouse

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

isolation-characterization-dendritic-cells-macrophages-from-mouse

Research

JoVE Journal

Immunology and Infection

38.9K Views.  Emory University. Qui, dettaglio una metodologia per l'isolamento rapido delle cellule dendritiche di topo intestinali (DCS) e macrofagi. Caratterizzazione fenotipica di DC intestinali e macrofagi viene eseguita utilizzando multi-color analisi citometrica mentre magnetica arricchimento tallone seguita dalla selezione cellulare viene utilizzato per produrre le popolazioni di elevata purezza per gli studi funzionali.

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

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

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Isolamento di cellule dendritiche mieloidi e cellule epiteliali di timo umano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Cellule T Capture batteri da Transinfection da cellule dendritiche

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolamento e analisi citofluorimetrica delle cellule immunitarie dal cervello ischemica mouse

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Generazione e identificazione di GM-CSF Derivato alveolo-come macrofagi e dendritiche cellule da midollo osseo di topo

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Caratterizzazione delle derivate da monociti cellule dendritiche umane di Citometria a Flusso Imaging: un confronto tra due protocolli di isolamento dei monociti

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caratterizzazione fenotipica dei macrofagi dal rene di ratto mediante citometria di flusso

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Separazione delle cellule fluorescenza-attivato per la purificazione delle cellule dendritiche plasmacitoidi dal midollo osseo di topo

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.