Il finanziamento per questo progetto è stata fornita da un americano Society Scholar Award Cancer Research, (06-024-01-CSM di Charles J. Dimitroff), un National Institutes of Health / National Cancer Institute di sovvenzione (RO1CA118124 di Charles J. Dimitroff) e uno National Institutes of Health / National Institute of Arthritis e muscolo-scheletrico e malattie della pelle di sovvenzione (P30 AR042689 il Dr. Thomas S. Kupper, Brigham and Women Hospital, Boston, MA).

1. Preparazione di hBMEC (HBMEC-60 Cell) monostrato in piastre di Petri per camera di flusso <ol><li> Cappotto 35 x 10 millimetri di coltura tissulare piatti con 2 ml di fibronectina 20μg/ml (in PBS) notte a 4 ° C o per 3 ore a 37 ° C. </li><li> Aspirare soluzione fibronectina da piatti e aggiungere 1,5 x10-5 HBMEC 60 celle [Medium 199 con HEPES e glutammina, 10% FBS, 10% siero umano, 5 unità / ml di eparina, 1ng/ml umano ricombinante del fattore di crescita dei fibroblasti, 1% di penicillina- streptomicina] per il piatto e permettere loro di crescere fino a confluenza&gt; 90%. (Questo richiede 2 giorni) </li><li> Aspirare terreni di crescita e, in up-regolazione dell&#39;espressione E-selectina, aggiungere mezzo fresco con 50ng/mL IL-1β per 4-6 ore. Monostrati cellulari sono ora pronti per la laminazione del saggio. Per bloccare la funzione E-selectina, neutralizzanti anti-umano E-selectina Moab (clone BBIG-E4 (5D11)) possono essere aggiunti in 20μg/ml per ~ 1 ora a 37 ° C. </li></ol> 2. Preparazione di cellule progenitrici emopoietiche umane KG1a per camera di flusso <ol><li> Umane ematopoietiche cellule progenitrici KG1a sono cresciuto fino a confluenza (1x10 6 cellule / ml) in RPMI-1640 con la glutamina e il 10% FBS / 1% di penicillina-streptomicina e vengono aggiunti direttamente dal pallone per l&#39;uso in test di flusso da camera. </li><li> Numero di cellulare è calcolato utilizzando un emocitometro e poi le cellule sono risospese in 1 x 10 6 cellule / ml in HBSS con 10mM HEPES (H / H Buffer) e 2mM CaCl 2 </li><li> Le cellule sono conservati in ghiaccio fino al momento dell&#39;uso in test. </li></ol> 3. Preparazione del Microscopio, Parallel-Plate Chamber portata e di pompa a siringa (Figura 1) <img src="/files/ftp_upload/old/1009_2009_1_26_7/1009_figure1.png" alt="Figura 1" width="600" height="409" /> Figura 1. Illustrazione di Parallel-Plate Analisi dei flussi Camera. Microscopio invertito, Harvard pompa a siringa, computer / macchina fotografica e piatti paralleli camera di flusso sono collocati in una disposizione ottimale per l&#39;analisi cellulare efficiente e riproducibile. <ol><li> Accendere e fonte di luce per microscopio invertito e il potere di pompa a siringa e selezionare l&#39;obiettivo 10X sul microscopio. </li><li> Luogo 50mL tubo conico in porta e riempire con H / H e 2mM CaCl 2. </li><li> Mettere 5 ml provette di plastica in portaprovettoni sotto il tubo 50mL conica </li><li> Luogo siringa da 60 ml a pompa a siringa (Assicurarsi che sia lo stantuffo della siringa e del corpo sono entrambi protetti). </li><li> Allega 3-way rubinetto alla fine della siringa da 60 ml e attaccare la siringa 5 ml di 3-way rubinetto. </li><li> Posto in parallelo Camera apparato di flusso (GlycoTech, Inc.) sul palco microscopio con tubo di ingresso a destra, il tubo di uscita a sinistra e tubo a vuoto alla schiena. </li><li> Collegare il tubo a vuoto per vuoto e la valvola aperta per consentire apparato piastra parallela di aderire al palco. (Questo test se la gonna a vuoto sulla camera di flusso è a tenuta d&#39;aria.) Spegnere valvola dell&#39;aria. </li><li> Collegare il tubo linea di uscita (a sinistra del microscopio) a 3-way rubinetto. Apparecchio è ora pronto per l&#39;immissione sul HBMEC-60 piastre monostrato. </li><li> Luogo HBMEC-60 piastra monostrato e posto sul centro del microscopio. </li><li> Accendere il vuoto e la posizione dell&#39;apparato camera di flusso sopra la piastra in modo che la piastra monostrato si trova nel centro. </li><li> Abbassare delicatamente l&#39;apparato camera di flusso sul HBMEC-60 piastre monostrato e permettono camera apparato flusso di aspirazione verso il basso. (Non ci dovrebbero essere i suoni di aria che fuoriesce e, a questo punto potrebbe essere necessario applicare una pressione alla camera per comprimere la guarnizione di gomma.) </li><li> Una volta vuoto è stato stabilito, ingresso tubo posto (a destra) nel tubo 50mL conica contenente H / H con 2mM CaCl 2. </li><li> Aperto a 3 vie rubinetto per consentire il flusso all&#39;interno della siringa da 60 ml e la pompa posto in &quot;modalità pompa.&quot; </li><li> Per eliminare l&#39;aria da ingresso / uscita senza sollevare le cellule sulla piastra, utilizzare la pompa a siringa fissato a 2mL/minute per riempire il tubo di aspirazione, poi impostare rapidamente a 0,5 ml / minuto appena prima il liquido raggiunge l&#39;apparato. E &#39;imperativo per primo questo set con cautela ed evitare bolle d&#39;aria che alzerà il monostrato dal piatto. </li><li> Una volta fluido è visto spostare nella siringa da 60 ml, la camera di flusso e pompa a siringa è stato innescato e la camera di flusso è pronto per l&#39;uso. Questo set up verrà ripetuta per ogni corsa cella di input e per ogni nuova piastra necessario. </li></ol> 4. Acquisizione di leucociti Eventi di Rolling utilizzando elementi NIS <ol><li> Aprire NIS Elements sul desktop. </li><li> Premere l&#39;icona &quot;play&quot; per l&#39;immagine live. Esposizione automatica ti permetterà di vedere il piatto più semplice per ora e &#39;importante per la messa a fuoco. </li><li> Assicurarsi che microscopio si concentra correttamente sul monostrato cellulare. </li><li> Una volta concentrata, re-impostare l&#39;esposizione a 1 luce fotogrammi / secondo e regolare sul microscopio. L&#39;immagine dovrebbe comparire sul computer e non sarà più visibile nel mirino microscopio a causa del livello di scarsa illuminazione. Istogramma della luce può essere annuncioportato alla giusta rimuovere esposizione dello sfondo o migliorare la chiarezza delle cellule. </li><li> Clicca su bin 2X2 o Bin Live per ottenere un&#39;immagine che è l&#39;ideale per la registrazione di filmati </li><li> Clicca su &quot;macro&quot; sulla barra dei menu e selezionare &quot;scrivi a porta&quot;, selezionare la porta &quot;COM3&quot; e &quot;aperto&quot;. (Questo apre la porta a coordinarsi con la pompa a siringa. </li><li> Pipettare 1 ml di sospensione cellulare KG1a nella provetta 5 ml vicino alla linea di tubi d&#39;ingresso e poi posto tubi linea di ingresso al fondo della provetta. </li><li> Vai alla barra dei menu, cliccare su &quot;Capture&quot;, &quot;time-lapse acquisizione.&quot; </li><li> Un nuovo menu pop-up. Selezionare il secondo protocollo della durata di 210, questa è la durata del programma per la laminazione a HBMEC-60 celle. (Ogni fase è programmata per scattare una foto ogni secondo per tutta la durata del protocollo pompa. E &#39;inoltre configurato per inviare un comando tramite la porta COM3 per avviare la pompa.) In caso di time-lapse è interrotto, la pompa non si ferma da solo e deve essere ripristinato. Premere Annulla per tornare a vivere immagine. </li><li> Impostare la pompa a siringa a &quot;Modalità di programma&quot;, secondo il manuale di istruzioni. (Modalità di programmazione permette una programmazione manuale delle portate specifiche (ad esempio i livelli di stress di taglio) di essere impartito all&#39;interno della camera e dettare le interazioni delle cellule KG1a il monostrato HBMEC-60 cell.) Impostazioni per cellulari KG1a rotola su cellule hBMEC sono stati i seguenti 4,2 dyne / cm 2 per 45 s, 0,3 dyne / cm 2 per 60 s, ed aumenta gradualmente ogni 15 s ad un massimo di 4,2 dyne / cm 2 </li><li> Muro sforzo di taglio (W st) nella camera è stato calcolato secondo la seguente equazione: W st = 6μQ / a 2 b, dove μ è la viscosità stima dei media (0,0076 P), Q è la portata volumetrica in ml / s, una è l&#39;altezza del canale (spessore della guarnizione) in cm (0,03 cm) e b è la larghezza del canale in cm (0,5 cm). Le portate sono state regolate utilizzando la modalità programma della pompa a siringa. </li><li> Microscopio e computer sono ora pronti a catturare time-lapse fotografia. Premere il tasto &quot;Start Esegui&quot; sul computer per iniziare l&#39;acquisizione dati. Durante l&#39;esecuzione del programma, assicurarsi di aggiungere supporto per la provetta contenente le cellule KG1a al fine di mantenere l&#39;ingresso / uscita tubi pieni di media (Assicurarsi che non ci siano bolle d&#39;aria). </li></ol> 5. Dipendenza della Sialylation Terminale e Glicoproteina di superficie per E-selectina-mediato dalle cellule KG1a 5. Laminazione (Figura 2; Clip Video di 5,1-5,5) <ol><li> Saggio della cellula KG1a rotolamento sulla non-stimolata HBMEC-60 cellule. (Controllo negativo), le cellule KG1a sono stati preparati e infuse nella camera di flusso nel vivo non-IL-1β-stimolata HBMEC-60 come descritto sopra. </li><li> Saggio della cellula KG1a rotolamento su HBMEC-60 cellule pre-trattati con IL-1β. (E-selectina-dipendenza di controllo) KG1a cellule sono state preparate e infusa nella camera di flusso su vivere IL-1β-stimolata HBMEC-60 come descritto sopra. </li><li> Saggio della cellula KG1a rotolamento su HBMEC-60 cellule pre-trattati con IL-1β e poi neutralizzanti anti-umano E-selectina Moab. (E-selectina-dipendenza di controllo) KG1a cellule sono state preparate e infusa nella camera di flusso su vivere IL- 1β-stimolato HBMEC-60 pretrattati con neutralizzanti anti-umano E-selectina moab come descritto sopra. </li><li> Saggio della sialidasi-digerito cellule KG1a rotolamento su IL-1β-stimolata HBMEC-60. KG1a cellule pretrattate con 0.1U/ml Vibrio neuraminidasi Cholerae per 1 ora a 37 ° C sono stati preparati e infuse nella camera di flusso su vivere IL-1β-stimolata HBMEC-60 come descritto sopra. </li><li> Saggio della proteasi digerito cellule KG1a rotolamento su IL-1β-stimolata HBMEC-60. Cellule KG1a pretrattati con proteasi, bromelina 0.2U/ml per 1 ora a 37 ° C, sono stati preparati e infuse nella camera di flusso su vivere IL-1β-stimolata HBMEC-60 come descritto sopra. </li></ol> 6. Analisi di NIS-Elements-Catturato Cellule KG1a e grafico di Cell KG1a dati di Rolling <ol><li> Dopo la sequenza temporale viene acquisita, salvare i dati. </li><li> Vai alla scheda &quot;misura&quot; nella barra dei menu e selezionare &quot;definire soglia.&quot; Spostare le barre in modo che le cellule desiderate sono colorati in rosso. Selezionare &quot;tutte le immagini&quot; e poi &quot;Ok&quot;. L&#39;intero time-lapse sequenza dovrebbe ora essere modificato con visibile globuli rossi al rotolamento. </li><li> Vai su &quot;Misura&quot; e seleziona &quot;restrizioni&quot;. (Restrizioni consentire all&#39;utente di escludere gli oggetti thresholded che non rappresentano cellule di rotolamento. Questo è principalmente sfondo creato dal HBMEC-60 monostrato.) Selezionare &quot;area&quot; e min di ingresso e valori massimi per l&#39;area per le cellule di rotolamento. Ripetere questo passaggio per &quot;allungamento&quot; e &quot;circolarità&quot;. </li><li> Selezionare &quot;misura&quot; e selezionare &quot;creare binario utilizzando le restrizioni&quot;, e impostare i valori di restrizione per ottimizzare NIS-Elements software di riconoscimento delle cellule che rotola su monostrato di HBMEC-60 cellule. </li><li> Torna alla &quot;misura&quot;, selezionare &quot;i dati di campo&quot;, quindi selezionare &quot;Dati reset&quot;. Chiudere questo menu. </li><li> Torna a &quot;misura&quot; e seselezionare &quot;campo di scansione&quot;. </li><li> Aperto &quot;Misura&quot;, &quot;Campo dati&quot;, selezionare &quot;numero o oggetti&quot; e poi selezionare &quot;tutti i campi&quot; ed esportare dati in Microsoft Excel. </li><li> Selezionare &quot;dati chiari&quot; per preparare NIS-Elements acquisizione di cellule per la prossima raccolta dei dati. </li></ol> Risultati: <img src="/files/ftp_upload/old/1009_2009_1_26_8/1009_figure3.png" alt="Figura 3" width="600" height="382" /> Figura 3. Parallel-Plate Chamber Analisi del flusso di E-selectina-Binding attività sulle cellule KG1a. KG1a cellule trattate con sialidasi o proteasi sono stati analizzati per la laminazione efficienza su monostrati di HBMEC-60 cellule pre-trattati con IL-1β. Controllo negativo rotolamento esperimenti sono stati condotti anche attraverso l&#39;analisi delle cellule KG1a rotolamento sulla non-IL-1β-stimolata HBMEC -60 cellule o su IL-1β-stimolata HBMEC-60 cellule trattate con neutralizzanti anti-umano E-selectina Moab (5D11). Frequenze di laminazione delle cellule sono stati eseguiti in triplice copia ed analizzati con NIS-Elements. (* Significatività statistica p &lt;0,001) In questo video basato su report, abbiamo fornito una rappresentazione visiva di come analizzare i leucociti - le interazioni delle cellule endoteliali in condizioni fisiologiche sollecitazioni di taglio. In particolare, abbiamo analizzato il ruolo di E-selectina - E-selectina ligandi nella mediazione tethering leucociti e laminazione, un comportamento adesivo necessaria per l&#39;impegno dei secondaria attività più stabile legame attraverso i recettori, come le integrine e recettori acido ialuronico. Utilizzando il parallelo-piastra camera di flusso concepiti per essere utilizzati con un microscopio invertito, Harvard pompa a siringa, videocamera e computer / cellulare hardware di acquisizione, abbiamo condotto esperimenti in cui E-selectina ligando + cellule KG1a sono state usate come il nostro modello di leucociti e cellule hBMEC (HBMEC- 60) stimolati con citochine pro-infiammatorie, IL-1β, sono state usate come la nostra E-selectina + modello umano delle cellule endoteliali (1-6). Come riportato in precedenza, abbiamo osservato robusta cellula KG1a rotolamento su IL-1β-stimolata HBMEC-60 cellule in un range di livelli di stress di taglio in modo E-selectina-dipendente (Figura 3) (differenza statisticamente significativa se confrontata con cella KG1a rotola su non-IL-1β HBMEC-60 stimolate le cellule). Controlli negativi, che consisteva di cellule KG1a rotolamento sulla non-IL-1β-stimolata HBMEC-60 cellule o su IL-1β-stimolata HBEMC-60 cellule pretrattate con anti-umano E-selectina mAb, sono stati eseguiti in parallelo per dimostrare la dipendenza di stimolazione citochine ed E-selectina espressione per l&#39;attività di laminazione (Figura 3). Inoltre, l&#39;analisi laminazione di cellule KG1a pretrattate con Vibrio cholerae neuraminidasi (sialidasi) o con la bromelina, un non-specifico della proteasi noto per digerire E-selectina superficie ligando glicoproteina, ha sottolineato l&#39;importanza dei residui di acido sialico terminale e glicoproteina di superficie per l&#39;E-selectina ligando attività (2,3).

<ol>
	<li>Dimitroff, C. J., Kupper, T. S., Sackstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+leukocytic+migration+to+inflamed+skin+with+a+novel+fluorosugar+modifier+of+cutaneous+lymphocyte-associated+antigen.">Prevention of leukocytic migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen.</a> J. Clin. Invest. 112, 1008-1018 .</li><li>Dimitroff, C. J., Lechpammer, M., Long-Woodward, D., Kutok, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rolling+of+human+bone-metastatic+prostate+tumor+cells+on+human+bone+marrow+endothelium+under+shear+flow+is+mediated+by+E-selectin.">Rolling of human bone-metastatic prostate tumor cells on human bone marrow endothelium under shear flow is mediated by E-selectin.</a> Cancer Res. 64, 5261-5269 (2004).</li><li>Dimitroff, C. J., Descheny, L., Trujillo, N., Kim, R., Nguyen, V., Huang, W., Pienta, K. J., Kutok, J. L., Rubin, 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=Identification+of+leukocyte+E-selectin+ligands,+P-selectin+glycoprotein+ligand-1+and+E-selectin+ligand-1,+on+human+metastatic+prostate+tumor+cells.">Identification of leukocyte E-selectin ligands, P-selectin glycoprotein ligand-1 and E-selectin ligand-1, on human metastatic prostate tumor cells.</a> Cancer Res. 65, 5750-5760 (2005).</li><li>Descheny, L., Gainers, M. E., Walcheck, B., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ameliorating+Skin-Homing+Receptors+on+Malignant+T+cells+with+a+Fluorosugar+Analog+of+N-acetylglucosamine:+P-selectin+Ligand+is+a+More+Sensitive+Target+than+E-selectin+Ligand.">Ameliorating Skin-Homing Receptors on Malignant T cells with a Fluorosugar Analog of N-acetylglucosamine: P-selectin Ligand is a More Sensitive Target than E-selectin Ligand.</a> J Invest Dermatol. 126 (9), 2065-2073 (2006).</li><li>Gainers, M. E., Descheny, L., Barthel, S. B., Liu, L., Wurbel, M., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-homing+receptors+on+effector+leukocytes+are+differentially+sensitive+to+glyco-metabolic+antagonism+in+allergic+contact+dermatitis.">Skin-homing receptors on effector leukocytes are differentially sensitive to glyco-metabolic antagonism in allergic contact dermatitis.</a> J. Immunology. 179 (12), 8509-8518 (2007).</li><li>Rood, P. M., Calafat, J., von dem Borne, A. E., Gerritsen, W. R., van der Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+human+bone+marrow+endothelial+cells:+characterisation+of+new+cell+lines.">Immortalization of human bone marrow endothelial cells: characterisation of new cell lines.</a> Eur J Clin Invest. 30 (7), 618-629 (2000).</li></ol>

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Piatti paralleli analisi camera di flusso è un sistema specializzato in test in vitro utilizzati per lo studio dei leucociti - interazioni adesive endoteliale in condizioni di stress di taglio simili a quelli impartiti sulla superficie di un post-capillari venule. Come illustrato qui nella nostra presentazione visiva, dimostriamo il valore di questo sistema per indagare il ruolo di E-selectina - E-selectina ligandi nella mediazione tethering leucociti e rotolando sulla superficie dell&#39;endotelio microvascolare attivata. Ci rivelano anche l&#39;applicazione della proteasi, glicosidasi e trattamenti anticorpi blocco per chiarire il ruolo di E-selectina e dei suoi ligandi in questo sistema. Queste analisi sono altamente riproducibili e aiutare a formare una base sperimentale per lo studio di E-selectina - E-selectina funzione di legante in vivo. Oltre a studiare l&#39;adesione cellulare con monostrati di cellule microvascolari citochina-attivati ​​endoteliale (vena ombelicale hBMEC o umana o per assorbimento cutaneo delle cellule endoteliali microvascolari), le analisi possono essere eseguite utilizzando umana ricombinante purificata E-selectina o molecole chimeriche E-selectina-Ig come substrato per l&#39;adesione cellulare. Interazioni adesive altri mediato attraverso leucociti (L-)-selectina e delle piastrine (P)-selectina possono essere analizzati utilizzando la tecnologia a piatti paralleli camera di flusso. Variando il tipo di cellule monostrato / proteine ​​substrato o di leucociti ingresso / selectina-transfectant modelli cellulari, i ricercatori possono studiare il ruolo di queste interazioni in condizioni fisiologiche sollecitazioni di taglio.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>F6178-500ML</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>0.5 M EDTA</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15575-038</td>
<td>&nbsp;</td>
<tr>
<td>1M HEPES</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Parallel-Plate Flow Chamber Kit</td>
<td>&nbsp;</td>
<td>GlycoTech</td>
<td>31-001</td>
<td>&nbsp;</td>
<tr>
<td>PHD2000 Programmable Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD 2000</td>
<td>&nbsp;</td>
<tr>
<td>35x10mm Tissue Culture Dish</td>
<td>&nbsp;</td>
<td>BD FALCON</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin , from human plasma</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>86088-83-7 F2006-2MG</td>
<td>&nbsp;</td>
<tr>
<td>Interleukin-1_, Human; Recombinant</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>I9401-5UG</td>
<td>&nbsp;</td>
<tr>
<td>HBSS (Hank s Buffered Saline Solution)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14170</td>
<td>&nbsp;</td>
<tr>
<td>Medium 199 w/ HEPES & Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12320-039</td>
<td>&nbsp;</td>
<tr>
<td>Human Serum</td>
<td>&nbsp;</td>
<td>Innovative Research, Inc.</td>
<td>IPLA-SER1</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Abraxis Pharmaceutical Products</td>
<td>504011</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human fibroblast growth factor</td>
<td>&nbsp;</td>
<td>R&D Systems</td>
<td>233-FB</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Plastic Test tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352008</td>
<td>&nbsp;</td>
<tr>
<td>Anti-human E-selectin clone BBIG-E4(5D11)</td>
<td>&nbsp;</td>
<td>R&D Systems	</td>
<td>BBA16</td>
<td>&nbsp;</td>
<tr>
<td>Bromelain</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>B-4882</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
				 	
	

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analysis of physiologic e-selectin-mediated leukocyte rolling on microvascular endothelium

E-selectina è un tipo-1 proteina presente sulla membrana delle cellule endoteliali microvascolari che aiuta a iniziare il reclutamento dei leucociti circolanti nei tessuti cutanei, ossei e infiammato. E-selectina espressione è costitutiva sul microcircolo cutaneo e ossa ed è inducibile da citochine pro-infiammatorie, quali IL-1α / e TNF-α, il microvasi nei tessuti infiammati. Questo recettore lectina media deboli interazioni di legame con carboidrati contro-recettore ligandi sui leucociti circolanti, che si traduce in un comportamento caratteristico di rotolamento. Poiché queste interazioni precedere eventi adesivo più stabile e attività diapedesi, caratterizzazione dei leucociti attività di rotolamento e l&#39;identificazione dei leucociti E-selectina ligandi sono stati gli obiettivi principali degli studi di traffico dei leucociti e l&#39;infiammazione e lo sviluppo di terapie anti-infiammatorie (1-5) . L&#39;intento di questo rapporto è di fornire una visuale, descrizione completa della tecnologia più diffusa per lo studio di E-selectina E-selectina interazioni ligando in condizioni fisiologiche del flusso sanguigno. Il nostro laboratorio in collaborazione con il Centro di Ricerca Malattie della pelle Harvard utilizza un state-of-the-art a piatti paralleli apparato camera di flusso accompagnato da visualizzazione digitale e software di nuova registrazione, NIS-Elements. Questa tecnologia ci permette di analizzare gli eventi adesione in tempo reale per la visualizzazione sullo schermo così come l&#39;attività di registrazione in sequenza in un formato video. Parametri di adesione cellulare, come la frequenza di rotazione, la resistenza al taglio e rilegatura / tethering efficienza, sono calcolati con NIS-Elements, esportati in un foglio Excel e sottoposti ad analisi statistica. Nella dimostrazione qui presentati, abbiamo impiegato i piatti paralleli camera di flusso per indagare E-selectina-dipendente l&#39;attività dei leucociti che rotola su cellule di midollo osseo umano vivente endoteliale (hBMEC). Umane ematopoietiche cellule progenitrici KG1a, che esprimono un alto livello di E-selectina ligando, sono stati utilizzati come modello dei leucociti, mentre un immortalata linea cellulare hBMEC, HBMEC-60 cellule, è stato utilizzato come modello delle cellule endoteliali (6). Per indurre e simulare nativo E-selectina espressione nella camera di flusso, HBMEC-60 cellule sono stati attivati ​​con IL-1. La nostra presentazione video ha mostrato che a piatti paralleli analisi del flusso è un metodo adeguato per lo studio fisiologico E-selectina-mediata dei leucociti al rotolamento e le attività che la caratterizzazione funzionale dei leucociti E-selectina ligando (s) nella camera di flusso può essere accertata mediante l&#39;implementazione della proteasi o glicosidasi digestioni.

Watch this Scientific Journal Video about Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium at JoVE.com

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

Questo rapporto fornisce una rappresentazione visiva di piatti paralleli analisi del flusso della camera per lo studio delle interazioni tra leucociti endoteliale sotto fisiologiche sollecitazioni di taglio. Questo metodo è particolarmente utile per indagare il ruolo di endoteliale (E)-selectina e leucociti E-selectina ligandi che leucociti innescare rotolamento sulla superficie delle cellule endoteliali.

Analisi di fisiologica E-selectina-mediata rotolamento dei leucociti sull&#39;endotelio microvascolari

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

analysis-physiologic-e-selectin-mediated-leukocyte-rolling-on

Research

JoVE Journal

Biology

19.0K Views.  Brigham and Women's Hospital. Questo rapporto fornisce una rappresentazione visiva di piatti paralleli analisi del flusso della camera per lo studio delle interazioni tra leucociti endoteliale sotto fisiologiche sollecitazioni di taglio. Questo metodo è particolarmente utile per indagare il ruolo di endoteliale (E)-selectina e leucociti E-selectina ligandi che leucociti innescare rotolamento sulla superficie delle cellule endoteliali.

Video: Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Ricerca

Biologia

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioingegneria reti umane in topi immunodeficienti microvascolari

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq Analisi trascrittomi in trombina-trattati e di controllo dell&#39;uomo polmonari cellule endoteliali microvascolari

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

Isolamento di microvascolare endoteliale tubi da arterie di resistenza mouse

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Misura e Analisi di extracellulare Acido prodotte per determinare glicolitica Tasso

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Espressione di proteine di fusione fluorescenti in macrofagi e cellule dendritiche derivate da midollo osseo Murine

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Misurazioni simultanee di calcio intracellulare e potenziale di membrana nell'endotelio cerebrale Mouse appena isolato ed intatto

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Un test di germinazione coroide ex vivo di angiogenesi microvascolare oculare

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Analisi della funzione E3 Ubiquitina ligasi

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.