Name: lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture
Uploaded: 2016-04-08T13:00:00.000+00:00
Duration: 10 min 31 s
Description: Watch this Scientific Journal Video about Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture at JoVE.com

biopsie cutanee da pazienti affetti da psoriasi sono stati gentilmente forniti dal Dr. Andreas Koerber (dipartimento di Dermatologia presso l&#39;Università di Essen, Germania) dopo il consenso orale o scritta informato per uso scientifico. XH è anche supportato da NSFC 61.263.039 e 11.101.321 NSFC.

NOTA: Le biopsie cutanee da individui sani sono stati ottenuti da addominale avanzi pelle di individui sottoposti a chirurgia plastica elettiva dopo consenso informato orale o scritta per uso scientifico. L&#39;uso di pelle umana è stato approvato e in conformità con le norme stabilite dai comitati etici medici per la ricerca umana del centro medico dell&#39;Università Radboud, Nijmegen, Paesi Bassi e dell&#39;Università di Essen, in Germania. 1. Preparazione di sospensioni singola cella dalla pelle umana (lavoro sterile in un flusso-armadietto se successiva coltura cellulare è obbligatorio) <ol><li> Preparare terreno di coltura cellulare: RPMI 1640 + penicillina / streptomicina (concentrazioni finali 100 unità / ml e 100 mg / ml, rispettivamente) + piruvato (0,02 mm) e Glutamax (0,02 mm), senza siero aggiunto. </li><li> Preparare terreno di coltura completo: terreno di coltura preparato al punto 1.1 + 10% di siero umano pool (HPS); Conservare a 4 ° C. Portate medie a 20 ° C ± 2 prima di noiing. </li><li> Ottenere la biopsia della pelle utilizzando un round strumento biopsia pugno 4 mm e tenerlo in RPMI1640 terreno di coltura completo a 20 ° C ± 2 per un massimo di 4 ore oppure a 4 ° C ON. Elaborare la biopsia appena possibile all&#39;arrivo in laboratorio. NOTA: più lunga conservazione di pelle influenzerà il rendimento delle cellule e la vitalità delle cellule. </li><li> Etichettare un tubo blu-capped dissociazione e aggiungere 5 ml di terreno di coltura completo nel tubo etichettati. </li><li> Aggiungere 2 ml di terreno di coltura completo in ogni pozzetto (in totale 3 pozzi) di una piastra sterile cultura 6-bene. Utilizzare strumenti sterili per posizionare la biopsia in un singolo pozzetto, lavare, spostarlo verso un secondo pozzo e ripetere questo passo ancora una volta, ottenendo così un totale di tre risciacqui. </li><li> Trasferire la biopsia cutanea ben sciacquati ad una piastra di Petri sterile, aggiungere 100 ml di mezzo di coltura completo sulla parte superiore della biopsia, e con attenzione raschiare il tessuto adiposo sottocutaneo utilizzando un bisturi sterile monouso acciaio inox. NOTA: Thè è un passaggio fondamentale. </li><li> Tagliare ogni biopsia cutanea in 4 pezzi più piccoli su una piastra di Petri sterile. campioni di trasferimento (fino a quattro di biopsie di 4 mm per tubo) al tubo di dissociazione preparato contenente 5 ml di terreno di coltura completo. </li><li> Chiudere bene le provette con il tappo, e collegare testa in giù per la manica del dissociatore tessuti automatizzato. Assicurarsi che tutto il materiale di esempio si trova nella zona del rotore. </li><li> Avviare il processo di dissociazione eseguendo il &quot;programma di m_spleen _01&quot; (un programma predefinito fornito dalla memoria interna dello strumento o tramite la carta del programma accompagnati) per dissociare la biopsia alla velocità di rotazione appropriato per 56 sec. </li><li> Dopo l&#39;elaborazione, staccare il tubo di dissociazione dal dissociatore e assicurarsi che tutto il materiale dissociato viene raccolto sul fondo della provetta. </li><li> Aggiungere 150 microlitri collagenasi IA (80 mg / ml) nel tubo di dissociazione e incubare il campione in un bagno d&#39;acqua a 37° C per 60 min. Aggiungere 100 ml di DNasi I (5 MU / ml) nel tubo thedissociation, mescolare bene. NOTA: maggiore concentrazione di collagenasi o il tempo di incubazione più lungo altererà la vitalità delle cellule. </li><li> Fissare il tubo di dissociazione al manicotto del dissociatore tessuti automatizzati ed eseguire il &quot;programma di m_ milza _01&quot; dissociare la biopsia ancora una volta. </li><li> Inserire un 70 mM colino cella nylon sulla parte superiore di un tubo Falcon 50 ml. Applicare materiali campione dissociati a questo filtro per rimuovere cellule grumi di cellule / tessuti detriti. </li><li> Lavare setaccio cellule una volta con 5 ml di terreno di coltura completo. Centrifugare a 20 ° C ± 2, 450 xg per 10 minuti e aspirare il surnatante. </li><li> Ripetere la fase di lavaggio ancora una volta. pellet cellulari risospendere in 300 ml di terreno di coltura completo. cella singola sospensioni sono pronti per ulteriori analisi; continuare con protocolli per colture cellulari ex vivo o citometria a flusso di analisi. </li><li> In caso di ulteriore intracellular citochina colorazione, stimolare cellule con PMA (12,5 ng / ml), Ionomicina (500 ng / ml) e brefeldina a (5 mg / ml) per 4 ore a 37 ° C, 5% CO 2 incubatore per 4 ore prima di eseguire il flusso analisi di citometria. </li></ol> 2. Polyclonal attivazione delle cellule T della pelle-residente (Ex Vivo Cell Culture) <ol><li> Aliquota sospensioni monocellulari 100 pl (preparata al punto 1.15) in un piatto fondo rotondo a 96 pozzetti. NOTA: Per ciascuna mm biopsia 4 pelle, le sospensioni monocellulari acquisiti possono essere suddivisi in almeno due pozzetti di una piastra da 96 pozzetti. </li><li> Aggiungere anti-CD3 / anti-CD28 mAb microsfere rivestite (25.000 perle per bene), ricombinante citochina umana rIL-2 (concentrazione finale 25 U / ml) e rIL-1b (concentrazione finale 50 ng / ml). </li><li> Coprire la piastra di coltura con un coperchio piatto ben etichettato-, incubare 5% di CO 2, 100% di umidità, 37 ° C incubatore. Cambiare media quando il colore medio si trasforma in giallo. <br/&gt; Nota: Una colonia a cellule chiare si può osservare dal giorno 8 di coltura cellulare. </li></ol> 3. Citometria a flusso Analisi di primaria / coltura le cellule della pelle-Resident T <ol><li> Preparare FACS buffer: PBS + 0,2% di BSA. </li><li> Trasferire le cellule di interesse in una piastra a 96 pozzetti v-bottom. Centrifugare a 20 ° C ± 2, 450 xg per 2 minuti, e aspirare il surnatante. </li><li> Risospendere il pellet in 100 ml di PBS. Centrifugare a 20 ° C ± 2, 450 xg per 2 minuti, e aspirare il surnatante. </li><li> cellule macchia con 100 ml di eFluorescence780 preparati coniugati risolvibile colorante vitalità (1: 1000 diluizione utilizzando PBS) a 4 ° C per 30 min. Aggiungere 100 pl di tampone FACS, centrifugare a 20 ° C ± 2, 450 xg per 2 minuti, e aspirare il surnatante. </li><li> Scegliete il termine anticorpi monoclonali marcatore di superficie delle cellule, per esempio: CD45-BV421 (HI30, 1:20), CD3-ECD (UCHT1, 01:50), CD4-PC5.5 (1388,2, 1: 200), CD8-APC-AlexFluo700 (B9.11, 1: 400), CD14-FITC (TUK4, 01:50), CD19-APC-AlexFluo750 (13-119, 1:50), CD56-PE (MEM-188, 01:50), CD25-PeCy7 (BC96, 01:50), CD11c-PeCy7 (BU15, 1,10), e CD1c-APC (AD5-8E7, 1,10), preparare gli anticorpi monoclonali miscela utilizzando tampone FACS in base ad ogni fattore di diluizione mAb testato. Definire le impostazioni del cancello utilizzando il controllo isotipo di anticorpo insieme con il campione non macchia. </li><li> Aggiungere 25 ml di anticorpi monoclonali preparato miscela in ciascun pozzetto. Incubare 20 minuti a 20 ° C ± 2, proteggere dalla luce. </li><li> Aggiungere 100 pl di tampone FACS, centrifugare a 20 ° C ± 2, 450 xg per 2 minuti, e aspirare il surnatante. </li><li> Per i campioni che richiedono soltanto la superficie di colorazione delle cellule, continuare con il passo 3.16. </li><li> In caso di intracellulare colorazione Foxp3: <ol><li> Preparare fissazione e tampone permeabilizzazione miscelando una parte di concentrato con 3 parti di diluenti. </li><li> Preparare buffer di permeabilizzazione 1x: 1 parte di 10x permeabilizzazione tampone + 9 parti di H 2 O. sterilizzato </li></ol></li><li> pellet Risospendere in 100 ml di fissazione e permeabilitampone zione, mescolare bene e incubare a 4 ° C per 30 min. </li><li> Aggiungere 100 microlitri di buffer permeabilizzazione a ciascun pozzetto, centrifugare a 450 xg a temperatura ambiente per 2 minuti, e aspirare il surnatante. </li><li> Lavare le cellule con tampone permeabilizzazione ancora una volta. </li><li> Selezionare anticorpi monoclonali intracellulari necessario, ad esempio, Foxp3-eFluo450 (PCH101, 01:50), IL-17A-AlexFluo488 (eBio64DEC1, 01:50) e IFNγ-PeCy7 (4S.B3, 1: 400). Preparare l&#39;impasto mAbs utilizzando tampone permeabilizzazione contenente 2% di siero normale di ratto in base ad ogni fattore di diluizione mAb testato. Definire le impostazioni del cancello utilizzando il controllo isotipo di anticorpo insieme con il campione non macchia. </li><li> Aggiungere 20 ml di preparato MAB-composto in ogni pozzetto. Incubare a 4 ° C per 30 minuti, al riparo dalla luce. </li><li> Dopo 30 minuti di incubazione, aggiungere 100 ml di tampone permeabilizzazione in tutti i pozzetti. Centrifugare a 20 ° C ± 2, 450 xg per 2 minuti, e aspirare il surnatante. </li><li> Lavare le cellule con permeabilizzazione BuffeR ancora una volta. Risospendere il pellet in 110 ml di buffer di FACS, e sospensioni cellulari trasferimento in tubo microFACS. NOTA: Il campione è pronto per la misurazione della citometria a flusso a 10 colori. </li><li> In caso di numero di cellule esatta richiesta, aggiungere 10 ml di sospensione uniforme ben miscelato di fluorosfere in ciascun campione immediatamente prima di eseguire misurazioni di citometria a flusso. </li></ol>

<ol>
	<li>Clark, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vast+majority+of+CLA++T+cells+are+resident+in+normal+skin.">The vast majority of CLA+ T cells are resident in normal skin.</a> J Immunol. 176, 4431-4439 (2006).</li><li>Zaba, L. C., Fuentes-Duculan, J., Steinman, R. M., Krueger, J. G., Lowes, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+dermis+contains+distinct+populations+of+CD11c+BDCA-1++dendritic+cells+and+CD163+FXIIIA++macrophages.">Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages.</a> J Clin Invest. 117, 2517-2525 (2007).</li><li>Gebhardt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+T+cells+in+nonlymphoid+tissue+that+provide+enhanced+local+immunity+during+infection+with+herpes+simplex+virus.">Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus.</a> Nature Immunol. 10, 524-530 (2009).</li><li>Boyman, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+development+of+psoriasis+in+a+new+animal+model+shows+an+essential+role+for+resident+T+cells+and+tumor+necrosis+factor-alpha.">Spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-alpha.</a> J Exp Med. 199, 731-736 (2004).</li><li>Christophers, E., Mrowietz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+infiltrate+in+psoriasis.">The inflammatory infiltrate in psoriasis.</a> Clin Dermatol. 13, 131-135 (1995).</li><li>Jiang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+infection+generates+non-migratory+memory+CD8++T(RM)+cells+providing+global+skin+immunity.">Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity.</a> Nature. 483, 227-231 (2012).</li><li>Havran, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+diversity+of+T-cell+receptor+gamma-chain+expression+of+murine+Thy-1++dendritic+epidermal+cells+revealed+by+V+gamma+3-specific+monoclonal+antibody.">Limited diversity of T-cell receptor gamma-chain expression of murine Thy-1+ dendritic epidermal cells revealed by V gamma 3-specific monoclonal antibody.</a> Proc Natl Acad Sci U S A. 86, 4185-4189 (1989).</li><li>Campbell, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7+expression+and+memory+T+cell+diversity+in+humans.">CCR7 expression and memory T cell diversity in humans.</a> J Immunol. 166, 877-884 (2001).</li><li>He, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+PKC+in+human+T+cells+using+sotrastaurin+(AEB071)+preserves+regulatory+T+cells+and+prevents+IL-17+production.">Targeting PKC in human T cells using sotrastaurin (AEB071) preserves regulatory T cells and prevents IL-17 production.</a> J Invest Dermatol. 134, 975-983 (2014).</li><li>Clark, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+isolation+of+skin+resident+T+cells+from+normal+and+diseased+human+skin.">A novel method for the isolation of skin resident T cells from normal and diseased human skin.</a> J Invest Dermatol. 126, 1059-1070 (2006).</li><li>Clark, R. A., Kupper, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+and+dermal+fibroblasts+induce+proliferation+of+natural+regulatory+T+cells+isolated+from+human+skin.">IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin.</a> Blood. 109, 194-202 (2007).</li><li>Keijsers, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+induction+of+cutaneous+inflammation+results+in+the+accumulation+of+extracellular+trap-forming+neutrophils+expressing+RORgammat+and+IL-17.">In vivo induction of cutaneous inflammation results in the accumulation of extracellular trap-forming neutrophils expressing RORgammat and IL-17.</a> J Invest Dermatol. 134, 1276-1284 (2014).</li><li>Hybbinette, S., Bostrom, M., Lindberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+dissociation+of+keratinocytes+from+human+skin+biopsies+for+in+vitro+cell+propagation.">Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation.</a> Experimental dermatology. 8, 30-38 (1999).</li><li>Hennino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-infiltrating+CD8++T+cells+initiate+atopic+dermatitis+lesions.">Skin-infiltrating CD8+ T cells initiate atopic dermatitis lesions.</a> J Immunol. 178, 5571-5577 (2007).</li></ol>

The authors have nothing to disclose.

Qui, vi presentiamo un protocollo per isolare in modo efficace le cellule T della pelle residenti da biopsie cutanee umane. Il vantaggio di questo protocollo è l&#39;isolamento del numero relativamente elevato di linfociti vitali, ed esprimendo marcatori di superficie rilevanti. I sottoinsiemi di cellule identificate sono state: le cellule, CD11c + DCs CD4 + e cellule T CD8 + e Foxp3 + CD25 +. È importante sottolineare che, ex vivo coltura di cellule T de...

La pelle, come l&#39;interfaccia principale tra il corpo e l&#39;ambiente, fornisce la prima linea di difesa contro fisica esterna, chimiche e insulti biologici quali ferite, radiazioni ultraviolette e microrganismi. Pelle è composto da due vani principali, l&#39;epidermide e il derma, e contiene una varietà di cellule del sistema immunitario, tra cui le cellule di Langerhans, macrofagi, cellule dendritiche (DC), e circa 20 miliardi di cellule T di memoria, quasi il doppio del numero di presenti in tutto il volume di sangue 1 , 2. Un numero sempre crescente di dati supporta l&#39;idea che la pelle ha funzioni immunologiche essenziali, sia durante l&#39;omeostasi dei tessuti e in diverse condizioni patologiche. Le cellule immunitarie residenti nella cute normale si pensa di condurre immunosorveglianza 3 e hanno dimostrato di giocare un ruolo nello sviluppo di malattie infiammatorie come la psoriasi 4. Nella cute lesionata psoriasica, sia CD4 + e CD8 + infiltrati cellule T erano osserved ed è stato dimostrato che il rapporto tra il CD4 e CD8 varia a seconda dello stato di malattia 5. Tuttavia, queste popolazioni di cellule sono difficili da studiare perché le tecniche esistenti consentono l&#39;isolamento di solo poche cellule. Le tecniche attualmente largamente utilizzati per l&#39;isolamento delle cellule T da pelle umana combinano dissociazione pelle meccanica con trattamento enzimatico. Biopsie cutanee umane sono ampiamente tritata e incubate con enzimi come tripsina, collagenasi e / o EDTA 6-8. Considerando che la pelle è un tessuto barriera che è altamente resistente alle forze di trazione e disgregazione meccanica, i metodi stabiliti di isolamento delle cellule T prodotte pochissime cellule, e anche i numeri inferiori di cellule vitali, che rende ex vivo di coltura cellulare di queste popolazioni cellulari difficile e stimolante. Qui, riportiamo un metodo modificato per isolare i linfociti provenienti sia sano e coinvolto pelle umana psoriasica combinando Mechanla dissociazione iCal della pelle utilizzando un dissociatore tessuto automatizzato invece del metodo stabilito di ampiamente tritare, insieme a digestione enzimatica con collagenasi. sono stati osservati vari sottoinsiemi di cellule immunitarie vitali tra DC e cellule T dopo la preparazione di una cella singola sospensione. È importante sottolineare che l&#39;espressione dei marcatori di superficie CD3, CD4 e CD8 è stato ben conservato. Le cellule così preparate, sono pronte per l&#39;uso in colture cellulari ex vivo o analisi di citometria di flusso. Questo protocollo è stato impiegato con successo per l&#39;analisi di biopsie cutanee singoli (4 mm) derivati ​​dalla cute lesionale di pazienti affetti da psoriasi. I risultati hanno mostrato che le cellule T dei pazienti della pelle residente prodotte citochine infiammatorie come più IL-17 e IFNγ rispetto ai volontari sani 9.

<table><tbody><tr><td>Disposable Biopsy Punch</td><td>Kai Europe</td><td>BP-40F</td><td>4 mm</td></tr><tr><td>Disposable sterile scalpels</td><td>Dalhausen</td><td>1100000510</td><td></td></tr><tr><td>gentleMACS C tube</td><td>Miltenyi Biotech</td><td>130-093-237</td><td>Blue-capped, used as the dissociation tube.</td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotech</td><td>130-093-235</td><td>Automated tissue dissociator. Using the &quot;program spleen_01&quot; for dissociation of the skin biopsy.</td></tr><tr><td>Cell strainer&amp;nbsp;</td><td>BD</td><td>352350</td><td>70 &amp;micro;m nylon</td></tr><tr><td>96-well U-bottom plate</td><td>Greiner Bio-One</td><td>650180</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>22409-015</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Life Technologies</td><td>11360-039</td><td></td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Human Pooled Serum (HPS)</td><td>in house prepared</td><td></td><td></td></tr><tr><td>Collagenase I</td><td>Sigma-Aldrich</td><td>C2674</td><td>Type 1-A, suitable for cell culture</td></tr><tr><td>DNase I</td><td>Calbiochem</td><td>260913</td><td></td></tr><tr><td>PBS</td><td>B Braun</td><td>3623140</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A4503-500G</td><td></td></tr><tr><td>Fixable Viability Dye (FVD) APC-eFluo780</td><td>eBioscience</td><td>65-0865-18</td><td>Stain dead cells prior to cell fixation; dilute with PBS at 1:1,000</td></tr><tr><td>Fixation/Permeabilization Concentrate</td><td>eBioscience</td><td>00-5123-43</td><td></td></tr><tr><td>Fixation/Permeabilization Diluent</td><td>eBioscience</td><td>00-5223-56</td><td></td></tr><tr><td>Permeabilization Buffer (10x)</td><td>eBioscience</td><td>00-8333-56</td><td></td></tr><tr><td>BV421 Mouse anti-human CD45</td><td>BD</td><td>563879</td><td>Clone: HI30; dilution factor 1:50</td></tr><tr><td>FITC Mouse anti-human CD14</td><td>Dako</td><td>T0844</td><td>Clone: TUK4; dilution factor 1:50</td></tr><tr><td>PE Mouse anti-human CD56</td><td>Dako</td><td>R7127</td><td>Clone: MOC-1; dilution factor 1:50</td></tr><tr><td>ECD Mouse anti-human CD3</td><td>Beckman - Coulter</td><td>A07748</td><td>Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining.</td></tr><tr><td>PC5.5 Mouse anti-human CD4</td><td>Beckman - Coulter</td><td>B16491</td><td>Clone: 13B8.2; dilution factor 1:200</td></tr><tr><td>PeCy7 Mouse anti-human CD11c</td><td>Beckman - Coulter</td><td>A80249</td><td>Clone: BU15; dilution factor 1:50</td></tr><tr><td>APC Mouse anti-human CD1c</td><td>Miltenyi Biotech</td><td>130-090-903</td><td>Clone: AD5-8E7; dilution factor 1:10</td></tr><tr><td>APC-AlexFluo700 Mouse anti-human CD8</td><td>Beckman - Coulter</td><td>A66332</td><td>Clone: B9.11; dilution factor 1:400</td></tr><tr><td>APC-AlexFluo750 Mouse anti-human CD19</td><td>Beckman - Coulter</td><td>A94681</td><td>Clone: J3-119; dilution factor 1:50</td></tr><tr><td>PeCy7 Mouse anti-human CD25</td><td>eBioscience</td><td>AD5-8E7</td><td>Clone: BC96; dilution factor 1:50</td></tr><tr><td>&amp;nbsp;PE Rat anti-human CLA</td><td>Miltenyi Biotech</td><td>130-091-635</td><td>clone: HECA-452; dilution factor 1:</td></tr><tr><td>eFluo450 Rat anti-human Foxp3</td><td>eBIoscience</td><td>48-4776-42</td><td>Clone: PCH101; intracellular staining; dilution factor 1:50</td></tr><tr><td>AlexFluo488 Mouse anti-human IL-17A</td><td>eBioscience</td><td>53-7179-42</td><td>Clone: eBio64DEC17; intracellular staining; dilution factor 1:50</td></tr><tr><td>PeCy7 Mouse anti-human IFNg</td><td>eBioscience</td><td>25-7319-41</td><td>Clone: 4S.B3; intracellular staining; dilution factor 1:400</td></tr><tr><td>Flow-Count Fluorospheres</td><td>Beckman - Coulter</td><td>7547053</td><td>Counting beads, for flow cytometry</td></tr></tbody></table>

lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture

La pelle umana ha una importante funzione di barriera e contiene varie cellule del sistema immunitario che contribuiscono a omeostasi tissutale e la protezione da agenti patogeni. Come la pelle è relativamente facile da accesso, fornisce una piattaforma ideale per lo studio periferici meccanismi di regolazione del sistema immunitario. cellule residenti immunitario in buona salute immunosorveglianza condotta della pelle, ma anche svolgere un ruolo importante nello sviluppo di malattie infiammatorie della pelle, come la psoriasi. Nonostante intuizioni emergenti, la nostra comprensione della biologia di base varie malattie infiammatorie della pelle è ancora limitata. Vi è una necessità di una buona qualità popolazioni (singolo) di cellule isolate da campioni di pelle biopsiati. Finora, procedure di isolamento sono stati gravemente ostacolati dalla mancanza di ottenere un numero sufficiente di cellule vitali. Isolamento e successiva analisi sono stati colpiti dalla perdita di marcatori lignaggio cellule immunitarie, a causa dello stress meccanico e chimico causato dalle procedure di dissociazione correnti averesospensione singola cella. Qui, descriviamo un metodo modificato per isolare le cellule T sia da salutare e coinvolto pelle umana psoriasica combinando dissociazione pelle meccanico utilizzando un dissociatore tessuto automatizzato e il trattamento collagenasi. Questa metodologia conserva espressione della maggior parte dei marcatori lignaggio immunitario come CD4, CD8, Foxp3 e CD11c sulla preparazione di sospensioni di cellule singole. Sono mostrati esempi di isolamento successo + T CD4 cellule e successiva analisi fenotipica e funzionale.

Il protocollo presentato qui produrrà tra 2.200 ± 615 (media ± SEM, pelle di volontari sani) fino a 178.000 ± 760 (media ± SEM, cute lesionata dei pazienti affetti da psoriasi) linfociti vitali da pelle umana quando si utilizza un singolo 4 millimetri biopsia cutanea. Diversi tipi di CD45 + cellule sono state identificate in cella singola sospensioni derivate dalla pelle di individui sani, tra cui CD4 + T-cellule (~ 45%), CD8 + T-cellule (~ 30%), e CD11c...

Watch this Scientific Journal Video about Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture at JoVE.com

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 ...

lymphocyte-isolation-from-human-skin-for-phenotypic-analysis-ex-vivo

Research

JoVE Journal

Immunology and Infection

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

14.5K Views.  Radboud University Medical Centre. 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.

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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento linfociti T umani, la cultura e l&#39;analisi delle migrazioni In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Ricerca

Immunologia e infezioni

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Isolamento dei fibroblasti papillari e reticolari dalla pelle umana mediante selezione cellulare attivata a fluorescenza

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Genetica

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Un semplice e rapido protocollo non-enzimaticamente dissociare freschi tessuti umani per l&#39;analisi di linfociti infiltranti

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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 ...

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.

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.

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

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

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 ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Isolamento di cellule umane endoteliali linfatico da Multi-parametro fluorescenza-attivato cell sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Medicina

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e la coltura di cellule staminali epiteliali da pelle umana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Biologia

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 ...

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 ...

Linfociti Isolamento da Pelle umana per l'analisi fenotipica e

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Isolamento linfociti T umani, la cultura e l'analisi delle migrazioni In Vitro