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 border="0" cellpadding="0" cellspacing="0" width="843">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Name of Reagent/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:299px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch</td>
			<td>Kai Europe</td>
			<td>BP-40F</td>
			<td>4 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable sterile scalpels</td>
			<td>Dalhausen</td>
			<td>1100000510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS C tube</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td>Blue-capped, used as the dissociation tube.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:281px;">gentleMACS Dissociator</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
			<td style="width:299px;">Automated tissue dissociator. Using the &#34;program spleen_01&#34; for dissociation of the skin biopsy.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer&#160;</td>
			<td style="width:145px;">BD</td>
			<td style="width:119px;">352350</td>
			<td>70 &#181;m nylon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">96-well U-bottom plate</td>
			<td style="width:145px;">Greiner Bio-One</td>
			<td style="width:119px;">650180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Life Technologies</td>
			<td>22409-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">GlutaMAX</td>
			<td>Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:281px;">Human Pooled Serum (HPS)</td>
			<td style="width:145px;">in house prepared</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Collagenase I</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">C2674</td>
			<td>Type 1-A, suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">DNase I</td>
			<td style="width:145px;">Calbiochem</td>
			<td style="width:119px;">260913</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PBS</td>
			<td style="width:145px;">B Braun</td>
			<td style="width:119px;">3623140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BSA</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">A4503-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fixable Viability Dye (FVD) APC-eFluo780</td>
			<td>eBioscience</td>
			<td>65-0865-18</td>
			<td style="width:299px;">Stain dead cells prior to cell fixation; dilute with PBS at 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation/Permeabilization Concentrate</td>
			<td>eBioscience</td>
			<td>00-5123-43</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation/Permeabilization Diluent</td>
			<td>eBioscience</td>
			<td>00-5223-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permeabilization Buffer (10x)</td>
			<td>eBioscience</td>
			<td>00-8333-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BV421 Mouse anti-human CD45</td>
			<td style="width:145px;">BD</td>
			<td style="width:119px;">563879</td>
			<td>Clone: HI30; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">FITC Mouse anti-human CD14</td>
			<td>Dako</td>
			<td style="width:119px;">T0844</td>
			<td>Clone: TUK4; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PE Mouse anti-human CD56</td>
			<td style="width:145px;">Dako</td>
			<td style="width:119px;">R7127</td>
			<td>Clone: MOC-1; dilution factor 1:50</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:281px;">ECD Mouse anti-human CD3</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A07748</td>
			<td style="width:299px;">Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PC5.5 Mouse anti-human CD4</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">B16491</td>
			<td>Clone: 13B8.2; dilution factor 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PeCy7 Mouse anti-human CD11c</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A80249</td>
			<td>Clone: BU15; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">APC Mouse anti-human CD1c</td>
			<td style="width:145px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-090-903</td>
			<td>Clone: AD5-8E7; dilution factor 1:10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">APC-AlexFluo700 Mouse anti-human CD8</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A66332</td>
			<td>Clone: B9.11; dilution factor 1:400</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:281px;">APC-AlexFluo750 Mouse anti-human CD19</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A94681</td>
			<td>Clone: J3-119; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PeCy7 Mouse anti-human CD25</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">AD5-8E7</td>
			<td>Clone: BC96; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">&#160;PE Rat anti-human CLA</td>
			<td style="width:145px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-091-635</td>
			<td>clone: HECA-452; dilution factor 1:</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">eFluo450 Rat anti-human Foxp3</td>
			<td>eBIoscience</td>
			<td>48-4776-42</td>
			<td style="width:299px;">Clone: PCH101; intracellular staining; dilution factor 1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">AlexFluo488 Mouse anti-human IL-17A</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">53-7179-42</td>
			<td style="width:299px;">Clone: eBio64DEC17; intracellular staining; dilution factor 1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">PeCy7 Mouse anti-human IFNg</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">25-7319-41</td>
			<td style="width:299px;">Clone: 4S.B3; intracellular staining; dilution factor 1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Flow-Count Fluorospheres</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td align="right" style="width:119px;">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

14.4K 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

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA-Ig celle in base Antigen Presenting artificiale per Efficiente Ex vivo Estensione dei diritti dell&#39;uomo e CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Valutare l&#39;innato rilevamento dell&#39;infezione da HIV-1 CD4 infetti<sup&gt; +</sup&gt; Cellule T dalle cellule dendritiche plasmacitoidi Utilizzando un<em&gt; Ex vivo</em&gt; Sistema di co-coltura.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, 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 ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Isolamento e<em&gt; Ex Vivo</em&gt; Cultura di Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Celle γδ T, un Extrathymic αβT-cellule progenitrici

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

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 ...

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 ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murino linfociti Etichettatura da<sup&gt; 64</sup&gt; Cu-anticorpo recettore Targeting per<em&gt; In Vivo</em&gt; Cell Traffico da PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Quantificazione del linfocita murino traffico In Vivo e chemiotassi del monocito umano In Vitro

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Valutazione della migrazione dei linfociti in un sistema di trasmigrazione Ex Vivo

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Linfociti Isolamento da Pelle umana per l'analisi fenotipica e

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Capitoli in questo video

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

HLA-Ig celle in base Antigen Presenting artificiale per Efficiente Ex vivo Estensione dei diritti dell'uomo e CTL

Valutare l'innato rilevamento dell'infezione da HIV-1 CD4 infetti

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

Isolamento e

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

Isolamento e analisi citofluorimetrica delle cellule immunitarie dal cervello ischemica mouse

Murino linfociti Etichettatura da

Quantificazione del linfocita murino traffico In Vivo e chemiotassi del monocito umano In Vitro

Valutazione della migrazione dei linfociti in un sistema di trasmigrazione Ex Vivo