Danilo Maddalo è un destinatario di una borsa di ricerca come Gruppo Young Investigator (YIG) presso l&#39;Istituto di tecnologia di Karlsruhe.

1. Semina le cellule <ol><li> Prima di iniziare, riscaldare il terreno di coltura, la PBS 1X e la tripsina a bagnomaria a 37 ˚ C </li><li> Prendete le cellule fuori dell&#39;incubatore e aspirare il terreno. </li><li> Applicare delicatamente 5 ml di PBS 1X sulle cellule di lavarli. </li><li> Aspirare il PBS 1X e applicare 1 ml di tripsina contenente 0,025% EDTA. </li><li> Ruotare delicatamente la piastra per avere una distribuzione uniforme della tripsina. </li><li> Mettere le cellule nell&#39;incubatore a 37 ˚ C per 5-10 minuti (a seconda del tipo di cellula). Di volta in volta verificare se le cellule sono staccati agitando il piatto. </li><li> Risospendere le cellule in 10-20 ml di medie e raccoglierle in un tubo di 50 ml falco. </li><li> Contare le celle utilizzando un contatore Beckman. Il numero di cellule dovrebbe essere sufficiente a garantire una confluenza di circa il 60-70%. </li><li> Piatto le cellule e permettere loro di semi per 6-8 ore. </li></ol> 2. Transperfezione <ol><li> Prima di transfezione, scaldare una aliquota del mezzo privo di siero in un bagnomaria a 37 ˚ C. </li><li> Portare la X-treme Gene 9 a temperatura ambiente. </li><li> Pipettare il medium siero libero in 2 tubi eppendorf (1 tubo = 1 trasfezione). </li><li> Preparare la X-treme Gene 9 nel siero attenzione di media liberi a pagamento che la punta non toccare la parete di plastica del Eppendorf e incubare per 5 minuti. Nel frattempo preparare due mix del DNA: in una provetta mescolare insieme i plasmidi per Renilla, lucciola e un controllo vettore vuoto, in un altro tubo Renilla, lucciola e il chaperone molecolare. </li><li> Aggiungere il mix di DNA per ogni eppendorf contenenti siero di media liberi + X-treme Gene 9, vortex e incubare a temperatura ambiente per 20 minuti. </li><li> Aggiungere goccia a goccia ogni mix su ogni piastra e incubare per 24 ore a 37 ° C e 5% CO 2 </li></ol> 3. Dividere le cellule <ol><li> 24 ore dopo la trasfezione, trypsinize le cellule, come descritto nella 1,4-1,6 e diluire ad una confluenza finale del 60-70% in un 6-pozzetti. </li><li> Preparare una piastra per la mancata scioccato di controllo e, come molti piatti come punti di tempo di recupero che devono essere analizzati, come mostrato in fig. 2. </li></ol> 4. Colpo di calore <ol><li> Per shock termico, pre-riscaldare siero contenente medie in un bagnomaria a 37 ˚ C. </li><li> Preparare &#39;buffer di calore shock&#39; diluendo nel siero cicloeximide supporto contenente una concentrazione finale di 20 mg / ml e MOPS a 20 mm. </li><li> Ora togliere i piatti dal termostato e aspirare il terreno. </li><li> Aggiungere ad ogni pozzetto 1 ml di &#39;tampone Colpo di calore&#39;, e incubare per 30 minuti a 37 ˚ C e 5% di CO 2 (comprende anche la piastra di riferimento in questo trattamento). Questo fermerà de novo sintesi proteica. </li><li> Nel frattempo lo switch su un bagnomaria e impostare il temperatura a 45 ˚ C. </li><li> Dopo l&#39;incubazione, sigillare il coperchio delle piastre con Parafilm precedentemente tagliati a strisce. La piastra di riferimento (100% del refolfed lucciola) viene lasciato in incubatrice. </li><li> Incubare le piastre a 45 ˚ C per 30 minuti. </li><li> Rimuovere le piastre dal bagnomaria, togliere il parafilm e mettere i piatti nuovo al incubatore per permettere il recupero. </li><li> Ad ogni vendemmia punto volta le cellule e lavarli in PBS 1X per centrifugazione a 2000Xg per 1 minuto a 4 ˚ C. </li></ol> 5. Lisi delle cellule e saggio luciferasi <ol><li> Per una lisi cellulare efficiente, scatto congelare il pellet di cellule in azoto liquido. </li><li> Aggiungere 200 ml di tampone di lisi passivo diluito 1:5 in acqua bidistillata in un tubo di vetro. </li><li> Aggiungere 30 ml di lisato di cellule in una piastra da 96 pozzetti. Ogni campione è pipettati in triplice copia. </li><li> Solo la &#39;non scioccato&#39; campioni di riferimento sarà utilizzato perleggere le attività Renilla luciferasi, in quanto questo passaggio viene utilizzato per verificare l&#39;efficienza pari trasfezione. </li><li> Preparare le soluzioni per l&#39;analisi. Per la soluzione diluita luciferina luciferina ad una concentrazione finale di 0,2 mM in Gly-Gly buffer. Per il buffer di reazione diluita DTT e ATP alla concentrazione finale di 1 mM Gly-Gly buffer. Per il buffer coelenterazine reazione, diluire coelenterazine ad una concentrazione finale di 0,2 mM in tampone coelenterazine. Tenere la soluzione e la reazione luciferina luce coelenterazine tampone protetta. </li><li> Ora la piastra a 96 pozzetti nel luminometro e avviare il programma &#39;Wallac 1420 Workstation&#39;. </li><li> Introdurre il Pump1 nella provetta contenente la soluzione falco coelenterazine. Chiudere il coperchio in modo da nessuna luce può entrare in contatto con il tubo. </li><li> Scegliete dal menu &#39;di manutenzione Dispenser&#39; e spuntare l&#39;opzione Pump1. </li><li> Selezionare l&#39;opzione &#39;Riempi&#39;. </li><li> Per modificare il layout della piastra di scegliere l&#39;opzione &#39;Esplora il protocollo ed i risultati&#39; dal menu. Selezionare il protocollo e doppio click su di esso. Ora selezionare i pozzi da leggere. </li><li> Torna alla &#39;Wallac Manager&#39; e fare clic su &#39;Start&#39;. La reazione tra il luciferasi Renilla e il suo substrato coelenterazine emette luce con lunghezza d&#39;onda di picco di 482 nm. </li><li> Dopo la lettura, andare su &#39;manutenzione Dispenser&#39; e selezionare &#39;vuoti&#39; e Pump1 di rilasciare tutti la soluzione residua nuovo al tubo. </li><li> Ora spostare il tubo in una provetta contenente acqua falco e spuntare &#39;Flush&#39;. </li><li> Dopo la fase di lavaggio, svuotare il tubo selezionando &#39;vuoto&#39;. </li><li> Posizionare il tubo dal Pump1 nel tubo tampone di reazione falco e quella da Pump2 nel tubo luciferina falco. </li><li> Modificare il layout della piastra. </li><li> Selezionare &#39;Riempi&#39; per Pump1 e Pump2 dal &#39;Dimenù Spenser Manutenzione &#39;. </li><li> Selezionare il protocollo e iniziare la lettura. La reazione tra il luciferasi Firefly e il suo substrato luciferina emette luce con lunghezza d&#39;onda di picco di 560 nm. </li><li> Al termine della lettura ripetere la procedura di Empty-Flush-vuoto per Pump1 e Pump2. </li><li> I risultati sono ora disponibili nel &#39;Wallac 1420 Explorer&#39;. </li><li> Ogni esperimento è associato ad un numero test, username, data di inizio e fine. </li><li> I risultati possono ora essere esportati come file Excel. </li><li> Per i calcoli, le cellule si dividono in tre gruppi: 1. non scioccato campioni di riferimento (insieme al 100% del ripiegato lucciola luciferasi), 2. cellule senza chaperone molecolare e calore scioccato, 3. cellule con chaperone molecolare e calore scioccato. </li></ol> 6. Rappresentante Risultati Ripiegamento delle proteine ​​è direttamente correlato al tempo di recupero quindi per verificare se il sistema funziona pro<html> Perly, un saggio deve essere effettuata la raccolta delle cellule in diversi momenti. Una diretta correlazione tempo / percentuale di ripiegamento indica che l&#39;esperimento è stato eseguito correttamente e rappresenta quindi un fattore limitante. Tuttavia va sempre considerato che il ripiegamento dopo la scossa di calore è un evento saturare e quindi una corretta analisi andamento temporale deve essere eseguita prima di iniziare qualsiasi esperimento. <img src="/files/ftp_upload/3540/3540fig1.jpg" alt="Figura 1"/> Figura 1. Panoramica dell&#39;esperimento. Test in ripiegamento in vivo richiede 3 o 4 giorni per essere completata. Il giorno 1 le cellule vengono seminate e transfettate. Le cellule giorno seguente sono suddivisi in un formato più piccolo e il giorno 3 sono scioccati calore. A questo punto è possibile eseguire il test luciferasi subito dopo la raccolta delle cellule o per salvare il pellet a -80 ° C ed eseguire il test in un altro momento _content &quot;&gt; <img src="/files/ftp_upload/3540/3540fig2.jpg" alt="Figura 2"/> Figura 2. Suddivisione cellulare. Il giorno 2 celle sono divisi in un 6-pozzetti. Ogni trasfezione sarà diluito in un pozzo in modo da avere tutti i trasfezione sulla stessa piastra. Ogni piatto corrisponde ad un particolare tempo di recupero più un non-scioccato piastra di riferimento. <img src="/files/ftp_upload/3540/3540fig3.jpg" alt="Figura 3"/> Figura 3. Rappresentante buon risultato. A. Percentuale di ripiegamento misurata direttamente mediante attività lucciola luciferasi. Ogni grafico a barre rappresenta la percentuale di ripiegamento in assenza (barre nere) e in presenza (barre rosse) di un chaperone molecolare in diversi punti il ​​tempo di recupero. B. Correlazione tra tempo e ripiegamento in assenza (linea nera) e in presenza (linea rossa) di accompagnatore. I valori di r quadrato indicano buona correlazione. In Fig.3A è indicato un rappresentante, se i risultati sono stati raccolti cellule minuti 15, 30, 45, 60 e 120 dopo la scossa di calore in assenza (barre nere) e in presenza (barre rosse) di chaperone molecolare. Percentuale di luciferasi ripiegato aumenta con il tempo di recupero prolungato e con la transfezione del chaperone molecolare. La correlazione tra il ripiegamento e il tempo per il controllo (Fig. 3B, linea nera) e il chaperone molecolare transfettate cellule (Fig. 3B, linea rossa) viene visualizzato. <img src="/files/ftp_upload/3540/3540fig4.jpg" alt="Figura 4"/> Figura 4. Rappresentante risultato negativo. A. Percentuale di ripiegamento misurata direttamente mediante l&#39;attività luciferasi. Ogni grafico a barre rappresenta la percentuale di ripiegamento in assenza (barre nere) e in presenza (barre rosse) di un chaperone molecolare in diversi punti il ​​tempo di recupero. B. Correlazione tra tempo e ripiegamento in assenza ed in presenza (rosso l) ina di accompagnatore. I valori di r quadrato indicano correlazione negativa. Figura 4A. Evidenzia un risultato rappresentativo di un esperimento non eseguite correttamente. Sia il controllo (barre nere) e la molecolari chaperone transfettate cellule (barre rosse) non hanno mostrato un aumento del ripiegamento delle proteine ​​con il tempo. Inoltre, la trasfezione di chaperone molecolare non ha comportato un aumento di ripiegamento. Questa scarsa correlazione è confermata in fig. 4B che mostra un valore di R quadro di 0,6619 e 0,1882 rispettivamente per il controllo (linea nera) e la molecolari chaperone transfettate cellule (linea rossa).

<ol>
	<li>Bukau, B., Weissman, J., Horwich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperones+and+protein+quality+control.">Molecular chaperones and protein quality control.</a> Cell. 125, 443-443 (2006).</li><li>Ritossa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+the+heat+shock+response.">Discovery of the heat shock response.</a> Cell Stress Chaperones. 1, 97-97 (1996).</li><li>Hendrick, J. P., Hartl, F. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+functions+of+heat-shock+proteins.">Molecular chaperone functions of heat-shock proteins.</a> Annu. Rev. Biochem. 62, 349-349 (1993).</li><li>Fuqua, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+proteins+and+drug+resistance.">Heat shock proteins and drug resistance.</a> Breast. Cancer. Res. Treat. 32, 67-67 (1994).</li><li>Guzhova, I., Margulis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+chaperone+as+a+survival+factor+in+cell+pathology.">Hsp70 chaperone as a survival factor in cell pathology.</a> Int. Rev. Cytol. 254, 101-101 (2006).</li><li>Whitesell, L., Lindquist, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP90+and+the+chaperoning+of+cancer.">HSP90 and the chaperoning of cancer.</a> Nat. Rev. Cancer. 5, 761-761 (2005).</li><li>Konstantinopoulos, P. A., Papavassiliou, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=17-AAG:+mechanisms+of+antitumour+activity.">17-AAG: mechanisms of antitumour activity.</a> Expert. Opin. Investig. Drugs. 14, 1471-1471 (2005).</li><li>Galluzzi, L., Giordanetto, F., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+HSP70+for+cancer+therapy.">Targeting HSP70 for cancer therapy.</a> Mol. Cell. 36, 176-176 (2009).</li><li>Ozacmak, V. H., Barut, F., Ozacmak, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+provides+neuroprotection+by+reducing+oxidative+stress+and+HSP70+expression+during+chronic+cerebral+hypoperfusion+in+ovariectomized+rats.">Melatonin provides neuroprotection by reducing oxidative stress and HSP70 expression during chronic cerebral hypoperfusion in ovariectomized rats.</a> J. Pineal. Res. 47, 156-156 (2009).</li><li>Schroder, H., Langer, T., Hartl, F. U., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DnaK,+DnaJ+and+GrpE+form+a+cellular+chaperone+machinery+capable+of+repairing+heat-induced+protein+damage.">DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.</a> EMBO. J. 12, 4137-41 (1993).</li><li>Thulasiraman, V., Matts, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+geldanamycin+on+the+kinetics+of+chaperone-mediated+renaturation+of+firefly+luciferase+in+rabbit+reticulocyte+lysate.">Effect of geldanamycin on the kinetics of chaperone-mediated renaturation of firefly luciferase in rabbit reticulocyte lysate.</a> Biochemistry. 35, 13443-13443 (1996).</li><li>Howarth, 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=Hsp40+molecules+that+target+to+the+ubiquitin-proteasome+system+decrease+inclusion+formation+in+models+of+polyglutamine+disease.">Hsp40 molecules that target to the ubiquitin-proteasome system decrease inclusion formation in models of polyglutamine disease.</a> Mol. Ther. 15, 1110-1110 (2007).</li><li>Nollen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bag1+functions+in+vivo+as+a+negative+regulator+of+Hsp70+chaperone+activity.">Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity.</a> Mol. Cell. Biol. 20, 1083-1083 (2000).</li></ol>

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In questo lavoro un protocollo per misurare l&#39;attività intracellulare ripiegamento di chaperon molecolare è presentato. Il dosaggio intero può essere eseguita in 3 o 4 giorni come mostrato dalla visione d&#39;insieme in fig. 1. La robustezza e la linearità del segnale luminoso prodotto dalla lucciola e la luciferasi Renilla rappresentano una solida base per la riproducibilità del protocollo. La fase critica del saggio è la scelta di un ...

Per questo esperimento &#39;Luce Vector&#39; una forma luminometro Perkin Elmer &#39;1420 Controriforma luminescenza è stato utilizzato Programmi per il luminometro: Renilla: Pompa 1, 100 microlitri di iniezione Tampone di reazione: Pump1, 70 microlitri di iniezione Luciferina: Pump2, iniezione 30μl Per i buffer preparare queste soluzioni stock in acqua bidistillata: <ul style="list-style:none"><li> - 1M MgSO 4: 246,48 g MgSO 4 * 7H 2 O in 1 litro </li><li> - 0,5 M EGTA: 190,18 g dell&#39;EGTA in 1 litro </li><li> - 1M KH 2 PO 4: 136,09 g di KH 2 PO 4 in 1 litro </li><li> - 1M K2 HPO 4: 228,23 g di K 2 HPO 4 * 3H 2 O in 1 litro </li><li> - NaCl 5M: 292,2 g di NaCl in 1 litro </li><li> - 0,5 M EDTA: 186,12 g EDTA * 2H 2 O in 1 litro &nbsp;</li></ul> Gly-Gly-tampone: <ul style="list-style:none"><li> - 25mm Glycylglycin </li><li> - 15mm MgSO 4 </li><li> - 4mm EGTA </li></ul> Per 1 litro usare 3,3 g di Glycylglycin, 15 ml di una soluzione 1M MgSO4 e 8 ml di una soluzione 0,5 M EGTA. Risolvere in acqua, portare il pH a 7,8 e portare a un volume complessivo di 1 litro. Tampone di reazione per Renilla / Coelenterazine buffer: <ul style="list-style:none"><li> - &amp; Nbsp; 13,4 mM KH 2 PO 4 </li><li> - 86,6 mM KH 2 PO 4 </li><li> - 0,5 M di NaCl </li><li> - 1 mM EDTA </li></ul> Per 1 litro usare 13,4 ml di una soluzione 1M KH 2 PO 4 soluzione, 86,6 ml di una soluzione 1M K 2 HPO 4 soluzione, 100 ml di una soluzione di NaCl 5M e 2 ml di una soluzione 0,5 M EDTA. <table border="1"><tr><td></td><td> Azienda </td><td> Catalogo / Numero del prodotto: </td></tr><tr><td> DMEM (Dulbecco Modified Aquila Media 1X) </td><td> GIBCO </td><td> 41966029 </td></tr><tr><td> Oro siero fetale bovino </td><td> PAA </td><td> A15-151 </td></tr><tr><td> X-treme Gene 9 DNA Transfection Reagent </td><td> Roche </td><td&gt; 06365809001 </td></tr><tr><td> PBS Fosfato Dulbecco Buffered Saline 1X </td><td> GIBCO </td><td> 14190-094 </td></tr><tr><td> MOPS 3 - (N-Morpholino) propanesulfonic acido </td><td> SIGMA-ALDRICH </td><td> M1254 </td></tr><tr><td> Cicloesimide </td><td> SIGMA-ALDRICH </td><td> C7698 </td></tr><tr><td> Lysis Buffer passiva 5X </td><td> Promega </td><td> E194A </td></tr><tr><td> Glycylglycin EGTA MgSO 4 (MgSO 4 * 7H 2 O) </td><td> SIGMA-ALDRICH Roth Roth </td><td> G7278 3054,2 P027.1 </td></tr><tr><td> KH 2 PO 4 K 2 HPO 4 (K 2 HPO 4 * 3H 2 O) NaCl EDTA </td><td> Roth Roth Roth Roth </td><td> 3904,1 6878,2 3957,1 8043,1 </td></tr><tr><td> Luciferina Firefly </td><td> Biosynth </td><td> L8200 </td></tr><tr><td> Coelenterazine </td><td> Biosynth </td><td> C7000 </td></tr><tr><td> DTT (ditiotreitolo) </td><td> Roth </td><td> 6908,2 </td></tr><tr><td> ATP (adenosina trifosfato) </td><td> Roche </td><td> 10519987001 </td></tr></table>

intracellular refolding assay

Questo protocollo descrive un metodo per misurare l&#39;attività enzimatica di chaperon molecolare in una cella e sistemi basati su possibili effetti di composti con attività inibitoria / stimolante. Chaperon molecolari sono proteine ​​coinvolte nella regolazione di protein folding 1 e hanno un ruolo cruciale nel promuovere la sopravvivenza delle cellule su insulti dello stress come heat shock 2, la fame di nutrienti e l&#39;esposizione a sostanze chimiche / veleni 3. Per questo motivo accompagnatori si trovano ad essere coinvolti in eventi come lo sviluppo del tumore, chemioresistance delle cellule tumorali 4, nonché neurodegenerazione 5. Progettazione di piccole molecole in grado di inibire o stimolare l&#39;attività di questi enzimi è quindi una delle strategie più studiate per la terapia del cancro 7 e malattie neurodegenerative 9. Il test qui descritto offre la possibilità di misurare l&#39;attività ripiegamento di un chaperone molecolare particolare e per studiare l&#39;effetto di compounds sulla sua attività. In questo metodo il gene del chaperone molecolare indagato è transfettate con una codifica espressione vettore per il gene della luciferasi lucciola. E &#39;stato già descritto che denaturato luciferasi lucciola può essere ripiegata da chaperon molecolare 10,11. Come normalizzare il controllo transfezione, un vettore di codifica per il gene della luciferasi è Renilla transfettate. Tutti trasfezioni descritto in questo protocollo vengono eseguiti con X-treme Gene 11 (Roche) in cellule HEK-293. Nella prima fase, la sintesi proteica è inibita trattando le cellule con cicloesimide. Successivamente le proteine ​​svolgimento è indotto da shock termico a 45 ° C per 30 minuti. Dopo il recupero a 37 ° C, le proteine ​​sono ri-piegati nella loro conformazione attiva e l&#39;attività della luciferasi lucciola è usato come read-out: la luce più sarà prodotto, la proteina più avrà riguadagnato la conformazione originale. Cellule scioccato non-calore sono impostati come riferimento (100% del ripiegatoluciferasi).

Watch this Scientific Journal Video about Intracellular Refolding Assay at JoVE.com

Intracellular Refolding Assay

In questo protocollo un metodo per misurare ripiegamento delle proteine ​​intracellulari dopo shock termico è descritto. Questo metodo può essere utilizzato per studiare foldases come chaperon molecolari e la loro co-fattori o composti in grado di influenzare la loro attività. Attività Firefly luciferasi viene utilizzato come reporter per misurare l&#39;attività ripiegamento accompagnatore.

Ripiegamento di analisi intracellulari

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Research

JoVE Journal

Biology

14.1K Views.  Karlsruhe Institute of Technology. In questo protocollo un metodo per misurare ripiegamento delle proteine ​​intracellulari dopo shock termico è descritto. Questo metodo può essere utilizzato per studiare foldases come chaperon molecolari e la loro co-fattori o composti in grado di influenzare la loro attività. Attività Firefly luciferasi viene utilizzato come reporter per misurare l'attività ripiegamento accompagnatore.

Video: Intracellular Refolding Assay

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Misura Vicino membrana plasmatica e Global Dynamics calcio intracellulare negli astrociti

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Ricerca

Biologia

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Un metodo per l&#39;isolamento del mouse isole pancreatiche e intracellulare cAMP Determinazione

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Saggi accoppiati per il monitoraggio delle proteine ​​in ripiegamento<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Intravitale Microscopia per le strutture Imaging subcellulari in topi vivi Esprimere proteine ​​fluorescenti

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

Imaging il traffico intracellulare di APP con GFP fotoattivabile

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

Rilevazione di intracellulare genica in cellule vive di murini, umana e suina origine utilizzando nanoparticelle di fluorescenza marcata

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

Purificazione e ripiegando a amiloide fibrille di (sua)<sub&gt; 6</sub&gt; -tagged proteina ricombinante Shadoo Espresso come corpi di inclusione in<em&gt; E. coli</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Identificazione di segnale intracellulare eventi indotta in cellule vitali per interazione con le cellule vicine sottoposti apoptotica delle cellule morte

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Ottica pH quantificazione di intracellulare in Drosophila melanogaster anatomista Epithelia con pH indicatore fluorescente geneticamente codificato

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Lente-free Video microscopia per la dinamica e analisi quantitativa della coltura delle cellule aderenti

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...