Name: isolation of infiltrating leukocytes from mouse skin using enzymatic digest and gradient separation
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Description: Watch this Scientific Journal Video about Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation at JoVE.com

Il National Institutes of Health (NIH R15 NS067536-01A1 a DC), la National Association Vulvodinia (premio a DC), e Macalester College di sostenuto questo lavoro. CB ha ricevuto una borsa di studio dalla Macalester College di Beckman Scholars Program, finanziato dal Arnold e Mabel Beckman Foundation. BTF e TM sono supportati da JDRF 2-2011-662. Ringraziamo il Dr. Jason Schenkel e il Dr. Giuliana Lewis per un parere tecnico, e tutti i membri attuali e precedenti del laboratorio Chatterjea per il loro aiuto e sostegno.

Nota: i topi 8-12 settimane vecchia femmina ND4 svizzero Webster, convenzionalmente sono alloggiati con libero accesso a cibo e acqua, sono stati utilizzati per questi studi Il protocollo sperimentale utilizzato (B13S1) è stato approvato dal IACUC del Macalester College.. 1. Sensibilizzazione e sfida con Oxazolone <ol><li> Giorno 0 <ol><li> Preparare camera di anestesia con l&#39;aggiunta di 3 ml di isoflurano per asciugamani assorbenti posizionati sotto rete in fondo a un 4 L con coperchio vaso di vetro. Per i topi ND4 femminile usato qui, il tempo nella camera è 30-60 secondi fino a quando il soggetto è adeguatamente anestetizzato; ottimizzare la somministrazione di anestetici per utilizzarlo ceppo di topi. </li><li> Radere la schiena di ogni topo anestetizzato e affiancare con un trimmer peli di animali. </li></ol></li><li> Giorno 1 <ol><li> Effettuare una soluzione al 2% di 4-ethoxymethylene-2-fenil-2-ossazolin-5-one (Ox) in etanolo assoluto, e incubare a 50 ° C per 15 minuti su un piatto rotante in un incubatore. </li><li> Brevemente anestetizzaretopo, utilizzando anestesia isoflurano come descritto al punto 1.1.1 e applicare 100 ml di 2% bue alla schiena rasata in diverse applicazioni di serie di circa 20 ml ciascuno. Attendere 5-10 secondi tra le applicazioni di serie per la soluzione ad asciugare. </li><li> Fare attenzione per evitare di versare la soluzione Bue 2% sulle regioni diverse dalla schiena, e aspettare che la soluzione ad asciugare tra le applicazioni. </li></ol></li><li> Giorni 5-7 <ol><li> Fare una soluzione 1% di (Ox) in etanolo assoluto, e incubare a 50 ° C per 15 minuti su un piatto rotante in un incubatore. </li><li> Delicatamente ma con fermezza immobilizzare il mouse alla collottola della base del collo e la coda con l&#39;addome rivolto verso l&#39;alto e il collo leggermente inclinata verso il basso (simile a una presa per un&#39;iniezione intraperitoneale) e applicare 100 ml di 1% Bue al fianco rasato in diverse applicazioni di serie e il tempo per asciugare la pelle poi come descritto sopra. </li><li> Per i topi di controllo, applicare 100 ml di veicoli etanolo assoluto per il flan rasatoK. </li></ol></li></ol> 2. Fianco pelle Harvest <ol><li> Euthanize topi utilizzando anidride carbonica e l&#39;inalazione accise accuratamente la zona desiderata della pelle del fianco (~ 25 mm x 25 mm della cute) con 10 cm forbici chirurgiche. </li><li> Utilizzando una lama chirurgica tagliente pulito, raschiare via il grasso in eccesso e il tessuto connettivo della pelle del fianco. </li><li> Posizionare 3 pezzi di pelle fianco da 3 topi in una provetta conica da 50 ml contenente 10 ml di HBSS RT [con rosso fenolo, senza calcio o magnesio, 5 mM acido etilendiamminotetraacetico (EDTA), 10 mM 4- (2-idrossietil) -1- L&#39;acido piperazineethanesulfonic (HEPES), e siero fetale bovino al 10% (FBS)]. </li></ol> 3. Pre-digestione del tessuto di lavaggio <ol><li> Luogo tubo contenente frammenti di pelle su un piatto rotante per 30 minuti in un incubatore non gasati secco mantenuto a 37 ° C. </li><li> Vortex vigorosamente per 10 secondi al termine del periodo di incubazione. </li><li> Filtrare il contenuto del tubo attraverso un 70 microncolino per eliminare il tampone di lavaggio e raccogliere il tessuto. Un unico filtro può essere riutilizzato durante tutta la procedura in un gruppo di trattamento biologico. </li><li> Utilizzando un paio di pinze, di trasferire la pelle del fianco dal filtro in un nuovo tubo conico da 50 ml contenente 10 ml di fresco, RT HBSS media (contenenti EDTA, HEPES, e FBS come descritto sopra). </li><li> Con un paio di forbici chirurgiche sezionare immersi nel tubo 12,5 centimetri, tritare ogni pezzo di pelle del fianco in modo che il pezzo di tessuto media è di circa 2,2 mm x 2,2 millimetri nella zona. </li><li> Luogo tubo contenente frammenti di pelle su un piatto rotante per 30 minuti in un incubatore non gasati secco mantenuto a 37 ° C. </li><li> Vortex vigorosamente per 10 secondi alla fine dell&#39;incubazione </li></ol> 4. Collagenase Digestione di pelle <ol><li> Filtrare il contenuto del tubo attraverso un filtro 70 micron, raccogliere e trasferire i pezzi della pelle del fianco in una nuova provetta conica da 50 ml contenente 10 ml fresh, RT HBSS multimediale integrato con 0,7 mg / ml collagenasi D. </li><li> Tubo Luogo terreno contenente la digestione e frammenti di pelle su un piatto rotante per 30 minuti in un incubatore non gasati secco mantenuto a 37 ° C. </li><li> Contenuti vortice di tubo vigorosamente per 10 secondi al termine dell&#39;incubazione. </li><li> Filtro contenuto della provetta attraverso un filtro 70 micron in una nuova provetta da 50 ml; mantenere il flusso continuo contenente il tessuto digerito e gettare il filtro e affiancare i residui della pelle. </li><li> Lavare il flusso continuo in 50 ml di HBSS supporti, centrifugare per 5 minuti a 350 g, e decantare il surnatante. </li></ol> 5. Densità centrifugazione gradiente <ol><li> Usando una pipetta sierologica, aggiungere 3 ml di RT 67% gradiente di densità centrifugazione media in 1X tampone fosfato salino (PBS) in un nuovo tubo da 15 ml per ogni campione. </li><li> Risospendere il pellet di cellule in 5 ml RT 44% di densità supporti centrifugazione in gradiente in HBSS con rosso fenolo. </li><li> Gentiluomoly strato 5 ml di media centrifugazione in gradiente di densità 44% contenente le cellule sulla parte superiore del gradiente di densità centrifugazione campione supporti 67% utilizzando una pipetta sierologica. Assicurarsi di impostare la pipetta alla velocità più bassa, e posizionare la pipetta lungo la parete del tubo conico. Fare attenzione a non tremare, interrompere o invertire il gradiente. </li><li> Impostare l&#39;accelerazione sul valore più basso, disinserire il freno, e centrifugare il gradiente a 931 xg per 20 minuti a temperatura ambiente facendo in modo che la centrifuga è equilibrata. </li><li> Dopo la centrifugazione, rimuovere con attenzione il gradiente dalla centrifuga, e trasportare delicatamente per il banco di laboratorio tiene il tubo in posizione verticale. Utilizzando un 5 ml plastica trasferimento pipetta, rimuovere lo strato di cellule all&#39;interfaccia dei mezzi di centrifugazione in gradiente di densità 44% e il 67% e il trasferimento in una nuova provetta conica da 15 ml. Se l&#39;interfaccia non è visibile, è sufficiente rimuovere circa 2 ml di liquido al 67% -44% di confine. </li><li> Riempire il tubo conico contenente cellule dal interfaccia con il 2% FBS in 1X PBS freddo, le cellule centrifugare per 5 minuti a 350 xg, e decantare il surnatante. Nota: Questa è la prima volta che le cellule possono essere raffreddati / mantenuto freddo, in quanto mineralizzazione sono a 37 ° C e gradini multimediali gradiente di densità di centrifugazione sono a RT. Le celle possono essere risospese in 1: 1 Trypan blue diluizione e conteggi e informazioni redditività possono essere ottenute a questo punto. </li></ol> 6. Blocco e colorazione per analisi citofluorimetrica <ol><li> Blocco FcγRII / recettori III sulle cellule per prevenire non specifica colorazione anticorpi. Per gli esperimenti descritti, fare Master Mix anticorpo in PBS con il 2% FBS contenente diluizione 1: 100 di anticorpi non coniugato contro CD16 / 32 (2.4G2) per legare e recettori blocco FcγRII / III. </li><li> Incubare le cellule bloccate con le opportune Master Mix di anticorpi contro CD3ɛ (145-2C11), CD4 (RM4-5), CD8α (53-6,7), CD11b (M1 / 70), CD11c (N418), CD44 (IM7), CD45 ( 30-F11), CD45R(RA3-6B2), CD62L (MEL-14), e Ly-6G / Ly-6C (Gr-1) (RB6-8C5), nonché Brilliant Violet 510 vivo macchia / morti per identificare le cellule morte. Utilizzare tutti gli anticorpi sia alla diluizione raccomandata dai produttori o precedentemente ottimizzata dai ricercatori. Per gli esperimenti qui descritti, utilizzare tutti gli anticorpi a diluizione 1: 100. </li><li> Lavare le cellule e risospendere in 50 microlitri PBS 1X tampone colorazione con 2% FBS. Tenere in ghiaccio fino a quando i dati di fluorescenza possono essere acquisiti su un citofluorimetro. Nota: è importante avere i parametri di compensazione pre-ottimizzati con un tessuto linfoide e un antigene di superficie cellulare comune (come CD4 o CD8) prima acquisizione dei dati di fluorescenza. </li><li> Per aumentare il numero di cellule per il flusso downstream citometria acquisire dati da campioni interi di cellule colorate. </li></ol> 7. Analisi Citometria a flusso di dati utilizzando metodi standard per realizzare il procedura descritta di seguito <ol><li> In primo luogo, porta sulla regione dei linfociti del forward scatter(area) da scatter laterale (area) trama. </li><li> Quindi, selezionare singole cellule utilizzando la dispersione in avanti (larghezza) per scatter laterale (larghezza), al fine di evitare l&#39;analisi doppiette. Questo può anche essere realizzato con un altezza scatter in avanti per l&#39;altezza side scatter plot. </li><li> Poi, cancello sulla stirpe (CD11b, CD11c, e CD45R / B220) -negativa e CD3 positivi eventi, di escludere i macrofagi, le cellule cellule dendritiche, e B, rispettivamente, e confine analizza al compartimento delle cellule T, se l&#39;obiettivo è, come qui, per valutare infiltrante cellule T. </li><li> Avanti, porta in cellule vive che escludono il live macchia / morti. </li><li> Infine, cancello sulla popolazione di cellule (vedi rappresentante la sezione risultati). </li></ol>

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Caratterizzare cambiamenti nei leucociti pelle-residenti in modelli di roditori di malattie della pelle come la dermatite atopica o psoriasi è importante per capire i collegamenti meccanicistici tra afflusso di cellule infiammatorie e la patologia della malattia. Qui si descrive una tecnica economica per isolare leucociti dal tessuto della pelle con attrezzature di base trova nella maggior parte dei laboratori di ricerca biomedica. Questa tecnica relativamente rapida evita l&#39;uso di macchine dissociazione tissue cos...

Le condizioni della pelle che vanno da dermatite da contatto, eczema, psoriasi, cellulite, infezioni fungine e ascessi a tumori cutanei non-melanoma sono risultati essere tra le 50 malattie più diffuse a livello mondiale, e la quarta causa globale di malattie non mortali nel 2010 1. Di conseguenza, l&#39;indagine dei meccanismi molecolari e cellulari alla base diverse patologie della pelle è una zona necessaria e attiva di ricerca. Modelli di roditori sono stati notevolmente utili per la comprensione delle condizioni infiammatorie della pelle come la dermatite atopica 2, 3 psoriasi, o infezione da Staphylococcus aureus 4. Protocolli economici, efficienti e semplici per la digestione enzimatica del tessuto cutaneo mouse può fornire preparazioni di cellule che possono essere utilizzati per una varietà di applicazioni a valle per comprendere meglio la fisiopatologia di malattie della pelle. Qui, un metodo semplice ed economico è descritto per digerito enzimatico di pelle topotessuto e l&#39;isolamento di leucociti infiltranti pelle che può essere utilizzato per la coltura cellulare, in vivo trasferimento adottivo, analisi citofluorimetrica e smistamento o studi di espressione genica. L&#39;obiettivo generale di questa procedura è quello di preparare una sospensione singola cella di leucociti-pelle infiltrante con la vitalità delle cellule di alta minimizzando i costi tipicamente associati con kit di reagenti personalizzate e dissociatori meccanici. Esistenti tessuto cutaneo metodi di dissociazione 5-7 può provocare vitalità delle cellule e la superficie di integrità basso marcatore, o richiedere i kit di enzimi personalizzate e costose macchine di dissociazione dei tessuti 8-11. Mentre la digestione del tessuto cutaneo orecchio del mouse è ragionevolmente diffusa 12-13, digerire il tessuto della pelle altamente cheratinizzato (ad esempio, dal fianco) può risultare in preparazioni di cellule contaminate con grandi quantità di detriti non cellulare. In un recente studio, Zaid e colleghi hanno digerito la pelle del fianco del mouse per 90 minuti a 2,5 mg / ml dispasi, follocon a capo 45 min a 3 mg / ml di collagenasi 7. In un altro studio, questi ricercatori hanno utilizzato più incubazioni con una digestione combinato di 2,5 ore, compreso l&#39;uso di tripsina / EDTA, collagenasi III, e dispasi 5. L&#39;uso di tripsina non è raccomandato per la digestione enzimatica della pelle, come il trattamento con tripsina da diversi costruttori ha mostrato di influenzare misurabile l&#39;integrità delle proteine ​​di superficie cellulare su cellule di mammifero 14-15. Inoltre, dispasi può avere effetti significativi sulla capacità proliferativa delle cellule CD4 e CD8α T e influenzare l&#39;abbondanza superficie di almeno 20 molecole, tra cui comune T marcatori di attivazione delle cellule, quali CD62L 16. Altri protocolli utilizzano RPMI 1640 nel mezzo di digestione 6. Tuttavia, la presenza di Mg 2+ e Ca 2+ in RPMI può causare aggregazione vasta cella 17. Un protocollo ideale per la dissociazione di tessuto dovrebbe puntare per la vitalità delle cellule in alto, bassolivelli di aggregazione delle cellule, e il minimo danno al cellulare proteine ​​di superficie. Alta qualità preparazioni cellulari linfonodo stromali sono stati compiuti con protocolli che utilizzano enzimi incubazione più brevi, Ca 2+ e Mg 2+ media liberi, e di evitare tripsina e dispasi 18. Tuttavia, non sono state stabilite protocolli di questo tipo per la dissociazione di tutta la pelle del mouse. Qui, un protocollo è descritto a dissociarsi, isolare, e arricchire leucociti-pelle infiltrazione dalla pelle del mouse fianco allergene-sfidati. In breve, la pelle asportata è pre-incubato in soluzione salina bilanciata di Hank (HBSS) con siero fetale bovino 10% per 1 ora per ammorbidire i tessuti per la digestione e rimuovere il tessuto adiposo della pelle o morti in eccesso. Questa è seguita da una fase 30 min di digestione enzimatica con 0,7 mg / ml di collagenasi D. collagenasi D ha effetti minimi sulla densità di marcatori di superficie cellulare, e nessun effetto sulla proliferazione delle cellule T in vitro 16,18, rendendoparticolarmente adatto per applicazioni che coinvolgono la caratterizzazione delle proteine ​​di superficie. A seguito di digestione enzimatica, discontinua gradiente di densità centrifugazione è stato utilizzato per rimuovere le cellule epiteliali e detriti dalla cella singola sospensione e arricchire per le cellule ematopoietiche. È importante sottolineare che questo procedimento evita costosi cellulari magnetico reagenti separazione basati su colonne e macchine dissociazione tissutale 8-11, e può essere eseguita con attrezzature e materiali trovati in un laboratorio di base ricerca biomedica. Ecco questo protocollo è stato utilizzato per isolare leucociti dalla pelle fianco sfidato tre volte con il oxazolone aptene (bue) in precedenza sensibilizzati topi ND4 svizzeri (adattato da 19). Le cellule sono state analizzate utilizzando citometria a flusso multiparametrica. Questa tecnica ha prodotto una sospensione cellulare con detriti minima e&gt; 95% vitalità dei linfociti isolati che sono state analizzate mediante flusso multi-parametrica citometria a misurare l&#39;infiltrazione di linfociti T e neutrofili nella sk interessatoin.

<table><tbody><tr><td>HBSS with phenol red, without calcium, without magnesium, liquid</td><td>Sigma Aldrich</td><td>55021C</td><td>Keep sterile until day of usage.</td></tr><tr><td>HEPES, &amp;ge;99.5% (titration)</td><td>Sigma Aldrich</td><td>H3375</td><td></td></tr><tr><td>EDTA disodium salt solution</td><td>Sigma Aldrich</td><td>E7889</td><td>Keep sterile until day of usage.</td></tr><tr><td>Fetal Bovine Serum, USDA, Heat Inactivated, Premium Select</td><td>MidSci</td><td>S01520HI</td><td>Keep sterile until day of usage.</td></tr><tr><td>Percoll, pH 8.5-9.5 (25&amp;deg;C)</td><td>Sigma Aldrich</td><td>P1644</td><td>Keep sterile. Percoll needs to be made isotonic with sterile 10X PBS prior to use.</td></tr><tr><td>Trimmer Combo Kit</td><td>Kent Scientific</td><td>CL9990-1201&amp;nbsp;</td><td>Use the larger trimmer for shaving the flank and back.</td></tr><tr><td>4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one</td><td>Sigma Aldrich</td><td>E0753-10G</td><td>Dissolve in 100% EtOH at 50&amp;deg;C for 15 minutes on a rotating plate.</td></tr><tr><td>Purified anti-mouse CD16/CD32 (2.4G2)</td><td>Tonbo Biosciences</td><td>70-0161</td><td>Antibody used to block non-specific antibody binding during antibody staining for flow cytometry</td></tr><tr><td>anti-CD3&amp;epsilon;-BV650 (145-2C11)</td><td>Becton Dickinson</td><td>564378</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD4-APC-eFluor780 (RM4-5)</td><td>eBioscience</td><td>47-0042-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD8&amp;alpha;-BV785 (53-6.7)</td><td>BioLegend</td><td>100749</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD11b-eFluor450 (M1/70)</td><td>eBioscience</td><td>48-0112-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD11c-eFluor450 (N418)</td><td>eBioscience</td><td>48-0114-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD44-BV711 (IM7)</td><td>Becton Dickinson</td><td>563971</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD45-FITC (30-F11)</td><td>Tonbo Biosciences</td><td>35-0451-U025</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD45R-eFluor450 (RA3-6B2)</td><td>eBioscience</td><td>48-0452-80</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-CD62L-PerCP-Cy5.5 (MEL-14)</td><td>BioLegend</td><td>104431</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>anti-Ly-6G/Ly-6C (Gr-1)-PE (RB6-8C5)</td><td>BioLegend</td><td>108407</td><td>Antibody used to stain cells for flow cytometry</td></tr><tr><td>Ghost Dye-BV510</td><td>Tonbo Biosciences</td><td>13-0870-T100</td><td>Viability dye for flow cytometry</td></tr><tr><td>LSR Fortessa</td><td>Becton Dickinson</td><td>N/A</td><td>Cell analyzer (18 parameters)</td></tr><tr><td>Collagenase D from &lt;I&gt;Clostridium histolyticum&lt;/I&gt;</td><td>Roche Applied Science</td><td>11088858001</td><td>Aliquot lyophilized enzyme at 5 mg/ml in HBSS with phenol red, without calcium, and without magnesium into 1 mL aliquots.&amp;nbsp; Store immediately at -20&amp;deg;C for up to six months.</td></tr><tr><td>ND4 Swiss female mice</td><td>Harlan</td><td>0-32</td><td>8-12 weeks old; conventionally housed with free access to food and water and used according to Macalester College&#039;s IACUC guidelines.&amp;nbsp;</td></tr></tbody></table>

isolation of infiltrating leukocytes from mouse skin using enzymatic digest and gradient separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

Collagenasi D trattata splenociti presentano livelli simili di CD4 e CD8 α sulle cellule T rispetto ai controlli multimediali trattati In primo luogo, gli effetti potenziali di collagenasi D sulla frequenza e la superficie abbondanza di lignaggio e di attivazione marcatori su sottogruppi di cellule T sono stati valutati utilizzando tessuto linfoide secondario come controllo. Una sospensione di splenociti ottenuti da topi è stato...

Watch this Scientific Journal Video about Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation at JoVE.com

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Isolamento di leucociti infiltranti da mouse pelle utilizzando enzimatica Digest e Separazione Gradient

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

isolation-infiltrating-leukocytes-from-mouse-skin-using-enzymatic

Research

JoVE Journal

Immunology and Infection

21.2K Views.  Macalester College. This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols. 

Video: Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

Isolamento di Brain-infiltrazione Leucociti

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Ricerca

Immunologia e infezioni

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Isolamento dei linfociti da mouse mucosa del tratto genitale

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

Isolamento e colorazione dei Keratinociti della pelle del topo per l'analisi specifica del ciclo cellulare dell'espressione proteica cellulare da citometria di massa

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Biologia dello sviluppo

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Linfociti Isolamento da Pelle umana per l&#39;analisi fenotipica e<em&gt; Ex Vivo</em&gt; Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

Preparazione di cella singola sospensioni per analisi citofluorimetrica provenienti da diverse regioni della pelle del mouse

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Mieloide isolamento delle cellule da topo pelle e drenante linfonodi seguito intradermica immunizzazione con vivo attenuato<em&gt; Plasmodium</em&gt; sporozoiti

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolando i linfociti del sistema immunitario intestinale piccolo per Mouse

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Isolamento delle cellule mononucleari Di Lamina propria dal colon Murine utilizzando Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolamento delle cellule endoteliali dai capillari linfatici dermici murini

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Autore in primo piano: Isolamento basato su biglie magnetiche di cellule endoteliali linfatiche dermiche murine

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

Isolamento di cellule endoteliali linfatiche dermiche murine

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

Isolamento di leucociti infiltranti da mouse pelle utilizzando enzimatica Digest e Separazione Gradient

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Linfociti Isolamento da Pelle umana per l'analisi fenotipica e

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Isolando i linfociti del sistema immunitario intestinale piccolo per Mouse

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Isolamento di cellule endoteliali linfatiche dermiche murine