This work was supported by the Neurotrauma Research Program (Western Australia). This project is funded through the Road Trauma Trust Account, but does not reflect views or recommendations of the Road Safety Council.

1. ottico di calibrazione: misurazione della luce emessa <ol><li> Per preparare l&#39;apparato consegna luce, collegare una sorgente di luce a banda larga (ad esempio, o lampada Xenon tungsteno) ad un&#39;alimentazione adeguata. Posizionare una lente collimatrice di fronte alla sorgente di luce per produrre un fascio collimato di luce. Passare la luce attraverso un filtro di calore liquido per rimuovere la maggior parte del calore dal fascio di luce. A seconda dell&#39;applicazione, concentrare il fascio collimato al diaframma all&#39;ingresso di una guida di luce liquida, che prevede la consegna più flessibile della luce (ad es., In un incubatore). </li><li> All&#39;altra estremità della guida di luce liquida, posizionare un secondo collimatore e quindi un supporto per l&#39;interferenza e filtri di densità neutri. Controllare che questo accordo produce una macchia uniformemente illuminato di luce della lunghezza d&#39;onda e intensità desiderata. Nota: La distanza tra l&#39;estremità della guida di luce e il piano del campione può essere necessario variare a secondal&#39;area che deve essere illuminato, ma ricordate che la distanza fra l&#39;estremità delle guide di luce aumenta, l&#39;intensità della luce diminuirà. Nel presente studio, questa distanza è 14 centimetri e produce una sezione trasversale del fascio che illumina uniformemente un&#39;area ben 3x3 su una piastra a 96 pozzetti. </li><li> Assicurarsi che la luce diffusa dalla lampada e componenti ottici associati è in grado di raggiungere il campione. </li><li> Accendere il refrigeratore d&#39;acqua per il filtro di calore liquido e assicurarsi che ci sia lo scambio di acqua attraverso la camicia del filtro. Accendere la fonte di alimentazione della lampada e attendere almeno 5 minuti per la sorgente di luce a banda larga per stabilizzare. Nota: è richiesto l&#39;uso del dispositivo di raffreddamento dell&#39;acqua per evitare il surriscaldamento del dispositivo. </li><li> Selezionare un filtro di interferenza a banda stretta (di solito descritta dalla loro lunghezza d&#39;onda centrale, picco trasmittanza e la larghezza a metà altezza (FWHM) larghezza di banda) per generare la banda desiderata dell&#39;illuminazione. Nota: I filtri di interferenza a banda stretta utilizzati in questo studio sono stati 442 nm, 550 nm, 671 nm e 810 nm. </li><li> Misurare la luce prodotta dalla lampada al piano in cui il campione deve essere posizionato durante il trattamento. Misurare la luce utilizzando una sonda irraggiamento calibrato (collettore coseno) collegato ad un spettroradiometro adeguata, utilizzando software proprietario in base alle istruzioni del produttore. </li><li> Utilizza filtri di densità neutri per regolare l&#39;intensità della luce fino ad ottenere il risultato desiderato. Nota: Nel presente studio, l&#39;intensità della luce passata da ogni filtro di interferenza è regolata per fornire un&#39;uscita quantal uguali in ciascuna delle quattro bande d&#39;onda di trattamento. E &#39;importante controllare la taratura regolarmente, in quanto la potenza della lampada è soggetta a modifiche. Nota: Dopo la costituzione dell&#39;apparecchio, le cellule sono pronte per essere illuminata figura 1 è una rappresentazione del dispositivo consegna luce utilizzata in questo studio.. </li></ol><img alt="Figura 1"src = &quot;/ files / ftp_upload / 52221 / 52221fig1.jpg&quot; /&gt; Figura 1. Immagine dell&#39;apparato consegna luce. Illustrated sono la fonte di energia di luce, lampada allo xeno con alloggiamento, lente di collimazione, filtro per l&#39;acqua, l&#39;apertura d&#39;ingresso, guida di luce liquida, seconda lente di collimazione, porta filtro, telaio e trattamento opaco carta nera. Si noti che i filtri di lunghezza d&#39;onda e intensità banda stretta non sono mostrati. 2. Cella Preparazione <ol><li> Il metodo di consegna R / NIR-LT descritto può essere applicato a qualsiasi tipo di cellula o sistema modello in vitro; come tali le seguenti descrizioni di coltura cellulare sono generali, con tecniche consolidate alla cultura feocromocitoma (PC12), Müller (RMC1) e cellule della retina misti primarie. </li><li> Prima della semina delle cellule per il trattamento della luce e ossidativo test di stress, cultura immortalato cellule nel loro mezzi di crescita rispettiva contenente integratori adeguati (ad es., FBS, antibiotici) in flas T75ks fino 70-80% confluenti. </li><li> Staccare le cellule immortalati da fiaschi T75, utilizzando il metodo appropriato per il tipo di cellule da testare ad esempio., Tripsina. Se necessario, prima di procedere alla semina di cellule in 96 pozzetti piastre test, pozzetti della piastra dosaggio cappotto con 10 mcg / ml di poli-L-lisina per 1h (ad es., Per le cellule PC12) o 10 mcg / ml di poli-L-lisina per 1h seguita da un / N incubazione O con 10 mcg / ml laminina (ad es., per le cellule retiniche misti). </li><li> Centrifugare le cellule per la raccolta a seconda dei casi (ad es., 3 min a 405 g per PC12 o 10 min a 218 xg per le cellule RMC1), rimuovere il surnatante e risospendere in 8 ml di mezzi appropriati di coltura cellulare. </li><li> Se le cellule retiniche misti devono essere utilizzati, preparare da P0-5 cuccioli di ratto neonatale di digestione enzimatica (papaina) secondo le procedure stabilite 19 </li><li> Contare il numero di cellule vitali esclusi 0,4% (w / v) trypan colorante blu, utilizzando un emocitometro. </li><li> Regolare suc densità cellulareh che le cellule saranno circa il 70-80% confluenti dopo 24 o 48 ore in cultura. (Per cellule PC12 la densità di semina è 4,0 x 10 5 cellule vitali / ml, per le cellule RMC1, è di 2,5 x 10 5 cellule vitali / ml e per cellule retiniche misti è 8 x 10 5 cellule / ml vitali). Dopo l&#39;aggiunta di cellule o piastre a 96 pozzetti trasparente o nero (utilizzati per l&#39;H 2 O 2 o DCFH-DA assay rispettivamente), in 100 ml di mezzi di crescita appropriata, consentono piastra di sedersi su una superficie piana a 37 ° C, 5 % di CO 2 (o qualunque condizione siano appropriati per il tipo cellulare specifico) per almeno 24 ore per permettere l&#39;adesione cellulare. </li><li> Cellule culturali in mezzi di crescita negli appositi vassoi 96 e fino a quando non sono il 70-80% confluenti (24 ore per PC12 e cellule RMC1, 48h per le cellule retiniche misti). </li></ol> 3. Aggiunta di glutammato Stressor a Celle <ol><li> Preparare L-acido glutammico monosodico concentrazioni idrato sale stressanti di 0-10mm in appropriate crescere pienoterreni di coltura th. </li><li> Rimuovere i supporti dalle cellule e lavare delicatamente le cellule 3 volte con PBS (PC12) o HBSS (RMC1 e cellule retiniche miste). Dopo lavaggi, aggiungere media-glutammato contenente fino ad un volume totale di 100 microlitri / pozzetto. </li></ol> 4. Prima dosaggi di trattamento Luce <ol><li> Subito dopo l&#39;aggiunta di glutammato o di stress di scelta, esporre le cellule al trattamento della luce alle lunghezze d&#39;onda e intensità desiderati mettendo sotto l&#39;apparato consegna luce preparato. Assicurarsi di modificare l&#39;output quantal del fascio luminoso utilizzando le combinazioni con sede di filtri di densità neutri a seconda della lunghezza d&#39;onda di essere somministrata. Nota: Negli esperimenti attuali, esporre le cellule per il trattamento della luce per 3 min mantenere la temperatura appoggiando i 96 pozzetti su un pad termico C 37 o. Il tempo di trattamento può essere variato come richiesto. <ol><li> Mettere una carta nera opaca sotto le celle per evitare che la luce riflette in pozzi adiacenti nella 96-well vassoio designato per gli altri parametri di trattamento. </li></ol></li><li> Se il trattamento è desiderata per lunghezze di tempo più lunghi, aggiungere tampone 25mM Hepes per mantenere il pH del terreno di coltura cellulare o idealmente, posizionare le piastre apparecchi e di trattamento all&#39;interno di un incubatore con concentrazione di CO 2 regolato al 5% di CO 2, o condizioni che sono ottimali per il tipo di cellula specifico. </li><li> Dopo il completamento del trattamento di luce, collocare le celle su una superficie piana e incubare a 37 ° C e 5% CO 2 (o qualunque condizioni sono ottimali per il tipo di cellula) per 24h. Nota: Altri dosaggi di R / NIR-LT possono essere somministrati come sopra descritto, come desiderato. </li></ol> 5. finale Dosaggio del trattamento Luce e rilevamento di ROS <ol><li> Prima di eseguire la fase finale del trattamento luce, preparano i reagenti da utilizzare per il rilevamento ROS. Preparare tampone 50 mM citrato (pH6.0), Triton X100, e H 2 O 2 lavora reagente di rilevamentoSoluzione (1: 2: 97 10mM rapporto H 2 O 2 reagente soluzione madre di rilevamento; 10 U / ml di rafano perossidasi; citrato di sodio 50 mM (pH 6,0)). Preparare DCFH-DA ad una concentrazione finale di 100μM nelle matrici appropriate. Nota: il reagente DCFH-DA per le cellule PC12 è nei media RPMI, reattivo DCFH-DA per le cellule RMC1 è nei media DMEM. Si noti che i reagenti di rilevazione disponibili in commercio hanno sensibilità differenziali a specifiche ROS e reagenti devono essere scelti con attenzione per fornire le informazioni desiderate per una singola applicazione. </li><li> Somministrare trattamento di luce alle cellule, come descritto nel passaggio 4.1-4.1.2. Assicurarsi che le cellule vengono trattate con i desiderati fluenze / lunghezze d&#39;onda della luce. </li><li> Subito dopo il trattamento della luce, rimuovere i media-glutammato contenenti e lavare due volte con soluzione tampone adeguata (per cellule PC12, PBS è usato e per le cellule RMC1, viene utilizzato HBSS). Eseguire i saggi ROS sulle cellule come segue: <ol><li> H 2 O 2 </sub&gt; dosaggio: aggiungere 45 ml di tampone 50 mM citrato (pH 6,0) e 5 microlitri Triton X100 a ciascuno dei pozzetti. Agitare delicatamente le cellule in un agitatore orbitale per 30 sec e incubare a 37 ° C e 5% CO 2 per 15 min. Aggiungere 50 ml di H 2 O 2 soluzione di lavoro di rilevamento e incubare per 30 minuti a temperatura ambiente. Misurare la fluorescenza utilizzando un lettore di piastre con lunghezza d&#39;onda di eccitazione di 530nm e una lunghezza d&#39;onda di emissione di 480 nm. </li><li> DCF dosaggio: aggiungere 100 microlitri della soluzione 100 mM di DCFH-DA a ciascun pozzetto ed incubare per 30 min a 37 ° C e 5% CO 2. Rimuovere il supporto contenente 100 mM DCFH-DA e lavare con soluzione tampone appropriato (come descritto sopra) due volte. Aggiungere 90 ml di soluzione tampone e 10 microlitri Triton X100 a ciascuno dei pozzetti e agitare delicatamente su un agitatore orbitale per 30 secondi prima di 15 min di incubazione a 37 ° C. Misurare DCF fluorescenza ottenuti utilizzando un lettore di piastre con lunghezza d&#39;onda di eccitazione di 480 nm e una Wavel emissioneength di 530nm. </li><li> Espresso ROS valori relativi alla concentrazione proteica di cellule rimanenti nei pozzetti, utilizzando un kit colorimetrico per quantificare la concentrazione di proteine ​​secondo le istruzioni del fabbricante, con riferimento ad una curva standard per calcolare mg di proteina. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Abbiamo adattato con successo un sistema di erogazione precisa della luce e tarato per fornire un meccanismo per lo studio di ottimizzazione di R / NIR-LT in vitro. Lunghezza d&#39;onda e intensità di parametri R / NIR-LT sono in grado di essere manipolato accuratamente ed efficacemente usando questo sistema. Abbiamo stabilito che la luce il trattamento delle cellule non ha portato alla morte delle cellule, anche se ROS non sono stati ridotti alle lunghezze d&#39;onda e dosaggi consegnati, nei tipi cellulari t...

Le specie reattive dell&#39;ossigeno (ROS) sono necessari per una serie di trasduzione del segnale e le normali reazioni del metabolismo cellulare, inclusi quelli di neuroprotezione 1. Tuttavia, quando il meccanismo endogeno antiossidante sono in grado di controllare la produzione di ROS, le cellule possono soccombere allo stress ossidativo 2,3. Dopo lesioni al sistema nervoso centrale, gli aumenti legati in presenza di ROS e lo stress ossidativo sono pensati per svolgere un ruolo importante nella progressione del danno 4,5. Nonostante l&#39;ampio numero di strategie per attenuare lo stress ossidativo che sono stati valutati, non vi sono attualmente completamente efficaci, le strategie antiossidanti clinicamente rilevanti per attenuare la produzione di ROS e lo stress ossidativo associato in uso clinico dopo neurotrauma 6. Pertanto, l&#39;attenuazione dello stress ossidativo resta un obiettivo importante per l&#39;intervento terapeutico 7. Miglioramenti seguonoR ing / NIR-LT sono stati riportati in una vasta gamma di lesioni e malattie, tra cui la riduzione delle dimensioni dell&#39;infarto miocardico, le complicanze renali ed epatiche durante il diabete, degenerazione della retina, lesioni del sistema nervoso centrale e ictus 8, forse riducendo lo stress ossidativo. Con particolare riguardo alla lesione del sistema nervoso centrale, studi preclinici di efficacia della luce 670nm hanno mostrato effetti positivi in modelli di degenerazione retinica 9-11, lesioni del midollo spinale 12, morte neuronale 13. Sono stati condotti studi clinici per l&#39;età a secco degenerazione maculare e sono attualmente in corso per l&#39;ictus 14, ma i risultati di questi studi non appaiono promettenti, forse a causa di un errore di impiegare un trattamento efficace parametri 15. Come tale, R / NIR-LT non è stato ampiamente adottato come parte della pratica clinica normale neurotrauma, pur essendo facile da amministrare, non invasivo e trattamento relativamente poco costoso. Ostacoli alla traduzione clinica includono la mancanza di un clmeccanismo di azione e l&#39;assenza di un protocollo standardizzato efficace 16,17 trattamento precoce compreso. Letteratura attuale per quanto riguarda la terapia della luce rivela una pletora di variazione di parametri di trattamento per quanto riguarda le fonti di irradiazione (LED o laser), la lunghezza d&#39;onda (ad esempio, 630, 670, 780, 810, 830, 880, 904nm), dosi totali (joule di irraggiamento / unità di superficie), la durata (tempo di esposizione), tempistica (insulto pre- o post), la frequenza del trattamento e modalità di consegna (impulso o continua) 8. La variabilità dei parametri di trattamento tra gli studi rende difficile il confronto e ha contribuito allo scetticismo per quanto riguarda l&#39;efficacia 16. Pertanto, l&#39;ottimizzazione della R / NIR-LT è chiaramente necessario, con sistemi di coltura di cellule in grado di fornire il meccanismo di screening high-throughput necessario per confrontare le molteplici variabili. Tuttavia ci sono alcuni sistemi di illuminazione disponibili in commercio in grado di fornire la flessibilità e il controllo sufficiente sulle wavelength e l&#39;intensità di effettuare tali esperimenti di ottimizzazione. Commercialmente dispositivi disponibili LED sono generalmente in grado di fornire più lunghezze d&#39;onda o intensità, con conseguente investigatori impiegano più dispositivi LED di diversi produttori, che possono variare non solo in intensità, ma anche lo spettro di lunghezza d&#39;onda della luce emessa. Abbiamo affrontato questo problema utilizzando una sorgente di luce a banda larga Xenon filtrata attraverso filtri di interferenza a banda stretta, generando così una gamma di lunghezze d&#39;onda e fluenze di luce, consentendo chiudere, accurato controllo dei parametri di R / NIR-LT. È importante notare che la dose terapeutica del trattamento è definito dal numero di fotoni che interagiscono con il photoacceptor (cromoforo), che, nel caso di R / NIR-LT è postulata essere citocromo c ossidasi (COX) 18. Energia Photon erogata varia con lunghezza d&#39;onda; significato uguali dosi di energia a diverse lunghezze d&#39;onda sarà compregiato diverso numero di fotoni. Pertanto, la luce emessa dal dispositivo è stato calibrato per emettere un numero uguale di fotoni per ognuna delle lunghezze d&#39;onda scelte da testare. Abbiamo sviluppato un sistema che può essere utilizzato per fornire R / NIR-LT in un intervallo di lunghezze d&#39;onda e intensità di cellule in vitro e dimostrato la capacità di misurare gli effetti del trasporto di R / NIR-LT sulla produzione di ROS in cellule sottoposte a lo stress glutammato.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>OxiSelect Intracellular ROS Assay Kit (Green Fluorescence)</td>
			<td>Cell Biolabs</td>
			<td>STA-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed Reagent</td>
			<td>Molecular Probes</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>300 Watt Xenon Arc Lamp</td>
			<td>Newport Corporation</td>
			<td>6258</td>
			<td>Very intense light source, do not look directly into the lamp. Ensure there is sufficient cooling to the lamp whilst it is switched on</td>
		</tr>
		<tr>
			<td>USB4000-FL Fluorescence Spectrometer</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>CC-3-UV Cosine Corrector for Emission Collection</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#956;m diameter quartz fibre optic</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraSuite Spectroscopy Platform</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>2300 EnSpire Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td>Acute toxicity, wear gloves when handling.</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>142-47-2 (anhydrous)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pheochromocytoma rat adrenal medulla (PC12) cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI1640) Media</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
			<td>Gibco</td>
			<td>10082-147</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Horse Serum, New Zealand origin</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100 mM Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100X MEM Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Retinal Muller (rMC1) cells</td>
			<td>University of California, San Diego</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>75cm2 Flasks</td>
			<td>BD Biosciences</td>
			<td>B4-BE-353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>25988-63-0</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025-134</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>1X Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>165U Papain</td>
			<td>Worthington</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96 well plates, clear bottom, black</td>
			<td>Corning</td>
			<td>CLS3603-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Clear Polystyrene 96-Well Plates Untreated; Well shape: Round; Sterile.</td>
			<td>Costar</td>
			<td>07-200-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Seesaw Rocker</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Density Filter Paper (0.3)</td>
			<td>THORLABS</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>442nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FL441.6-10</td>
			<td></td>
		</tr>
		<tr>
			<td>550nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB550-10</td>
			<td></td>
		</tr>
		<tr>
			<td>670nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB670-10</td>
			<td></td>
		</tr>
		<tr>
			<td>810nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB810-10e</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.1)</td>
			<td>THORLABS</td>
			<td>NE01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.2)</td>
			<td>THORLABS</td>
			<td>NE02B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.3)</td>
			<td>THORLABS</td>
			<td>NE03B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.5)</td>
			<td>THORLABS</td>
			<td>NE05B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.6)</td>
			<td>THORLABS</td>
			<td>NE06B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (1.0)</td>
			<td>THORLABS</td>
			<td>NE10B</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro

Red / terapia della luce nel vicino infrarosso (R / NIR-LT), consegnato da laser o diodi emettitori di luce (LED), migliora i risultati funzionali e morfologiche a una serie di lesioni del sistema nervoso centrale in vivo, possibilmente riducendo lo stress ossidativo. Tuttavia, gli effetti di R / NIR-LT sullo stress ossidativo hanno dimostrato di variare a seconda lunghezza d&#39;onda o intensità di irraggiamento. Gli studi che confrontano parametri di trattamento mancano, a causa della mancanza di dispositivi disponibili in commercio in grado di offrire di più lunghezze d&#39;onda o intensità, adatti per alta through-put in studi di ottimizzazione in vitro. Questo protocollo descrive una tecnica per la consegna di luce in un intervallo di lunghezze d&#39;onda e intensità di ottimizzare dosi terapeutiche richieste per un determinato modello di lesione. Abbiamo ipotizzato che un metodo di fornire luce, in cui i parametri di lunghezza d&#39;onda e intensità potrebbero facilmente essere alterati, potrebbe facilitare la determinazione di una dose ottimale di R / NIR-LT per ridurre le specie reattive dell&#39;ossigeno(ROS) in vitro. Non coerente Xenon è stata filtrata attraverso filtri interferenziali a banda stretta per offrire lunghezze d&#39;onda diverse lunghezze d&#39;onda (centro di 440, 550, 670 e 810 nm) e fluenze (8,5 x 10 -3 a 3,8 x 10 -1 J / cm 2) della luce a cellule in coltura. L&#39;emissione luminosa dall&#39;apparato è stato calibrato per emettere terapeuticamente rilevanti, dosi uguali quantali di luce ad ogni lunghezza d&#39;onda. Specie reattive sono state rilevate in glutammato sottolineato cellule trattate con la luce, utilizzando DCFH-DA e H 2 O 2 coloranti fluorescenti sensibili. Abbiamo consegnato con successo luce in una gamma di lunghezze d&#39;onda fisiologicamente e terapeuticamente e intensità rilevanti, a cellule in coltura esposte a glutammato come modello di lesioni del sistema nervoso centrale. Mentre le influenze di R / NIR-LT utilizzati in questo studio non esercitare un effetto sulla ROS generato dalle cellule in coltura, il metodo di consegna luce è applicabile ad altri systems compresi mitocondri o più fisiologicamente rilevanti modelli cultura fetta organotipiche isolate, e potrebbe essere utilizzato per valutare gli effetti su una serie di misure di outcome del metabolismo ossidativo.

L&#39;uscita di luce emesso alla lunghezza d&#39;onda di 670nm è stato calibrato utilizzando filtri di densità neutri per irradiare le cellule con una gamma di fluenze che comprende una dose di luce 670nm precedentemente dimostrato di essere utile in vivo (0,3 J / cm 2) 20. Poiché il numero di filtri di densità neutri di fronte alla sorgente luminosa aumentata, l&#39;intensità (W / m 2) è diminuita, con meno luce che passa per l&#39;area di destinazione. Tabella 1 presenta i dati di ...

Watch this Scientific Journal Video about Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro at JoVE.com

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Metodo per la valutazione degli effetti di un intervallo di lunghezze d&#39;onda e intensità di / vicino-infrarosso di terapia della luce rossa sullo stress ossidativo<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

method-for-assessment-effects-range-wavelengths-intensities-rednear

Research

JoVE Journal

Biology

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

8.6K Views.  The University of Western Australia. Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Video: Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

Un test per misurare capacità di risposta comportamentale di Zebrafish a variazioni di intensità di luce

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Ricerca

Biologia

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Titolazione biochimica di glicogeno<em&gt; In vitro</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Analisi dello Stress Ossidativo in embrioni di zebrafish

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Misura Stress ossidativo Resistenza<em&gt; Caenorhabditis elegans</em&gt; In 96 pozzetti micropiastre

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

La quantificazione di adesività delle cellule endoteliali<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Spazi di giunzione intercellulare di comunicazione: un biomarker funzionale per valutare gli effetti avversi dei tossici e tossine, e benefici per la salute di prodotti naturali

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Analisi del trasferimento di gig Junction-dependent di miRNA con la microscopia 3D-FRAP

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Metodo per valutare gli effetti del batterioiocinio sul microbiota di gatti dei topi

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Indagando i dannosi effetti di basso pressione sterilizzazione al Plasma sulla sopravvivenza di Bacillus subtilis spore utilizzando Live Cell microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...