Name: visualization of bacterial toxin induced responses using live cell fluorescence microscopy
Uploaded: 2012-10-01T13:00:00
Description: Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Forfatterne vil gerne takke Richard Rest for den generøse gave anthrolysin O, Michael Caparon for den generøse gave af SLO plasmid og Jonathon Franks til teknisk bistand. Dette arbejde blev finansieret af NIH tilskud T32CA82084 (PAK), og R01AI072083 (RDS).

1. Oprensning af streptolysin O (SLO) <ol><li> Inokulere 20 ml overnatskultur af BL21 GOLD celler indeholdende pBADgIII-SLOhis plasmid 9 i 0,5 L LB-bouillon, og der tilsættes 500 pi 50 mg / ml ampicillin. Rystekultur ved 225 rpm ved 37 ° C indtil OD600 = 0,6, sædvanligvis ~ 1,5 timer. Centrifuger 1 ml bakterier (anset T 0) ved 10.000 xg 5 min, opløses pellet i 140 pi 1x SDS prøvepuffer / 1 OD og soniker at forskyde DNA til protein renhed analyse. </li><li> Inducere bakterier med t...

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Teknikkerne beskrevet her tillader undersøgelse af responser af immunceller til bakterietoksiner. Det mest kritiske trin er at håndtering og dosering af toksinet. Toksinaktivitet kan være yderst variable, selv mellem forskellige portioner af det samme præparat, på grund af dets skrøbelighed. Dette nødvendiggør enten prøvning af hver enkelt portion af toksin mod en reference cellelinje eller røde blodlegemer eller bruger toksin gradienter. Toxin gradienter, som leveret af mikropipette, tillade det fulde spektru...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> Ni-NTA agarose </td><td> Qiagen </td><td> 30.210 </td><td></td></tr><tr><td> polymixin-agarose </td><td> Sigma </td><td> P1411-5ML </td><td></td></tr><tr><td> Zeba afsalte Spin col </td><td> Fisher </td><td> PI-89891 </td><td></td></tr><tr><td> får RBC </td><td> Fisher </td><td> 50-415-688 </td><td></td></tr><tr><td> pBADgIII-SLO </td><td> N / A </td><td> N / A </td><td> se ref 9 </td></tr><tr><td> Cy5 monoreactive farvestof </td><td> GE Healthcare </td><td> PA25001 </td><td></td></tr><tr><td> Fura2-AM </td><td> Life Technologies </td><td> F1221 </td><td></td></tr><tr><td> Calcein AM / Ethidium homodimer </td><td> Life Technologies </td><td> L3224 </td><td></td></tr><tr><td> Anti-CD11c-APC </td><td> BD Biosciences </td><td> 550.261 </td><td></td></tr><tr><td> collagen-coated glas-bottom dish </td><td> Mattek </td><td> P35GCol-1,5-10-C </td><td></td></tr><tr><td> femto-tip II </td><td> Fisher </td><td> E5242957000 </td><td></td></tr><tr><td> Microloader </td><td> Fisher </td><td> E5242956003 </td><td></td></tr><tr><td> dextran Alexa 555 </td><td> Life Technologies </td><td> D34679 </td><td></td></tr><tr><td> Injectman Ni2 </td><td> Eppendorf </td><td> 920000029 </td><td></td></tr><tr><td> FemtoJet </td><td> Eppendorf </td><td> 5247 000.013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabel 1. Liste og kilde til specifikke reagenser og equipment nødvendig. Særligt udstyr og reagenser, der anvendes i denne protokol, sammen med virksomheden og katalognummer er opført. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Buffer </td><td> Sammensætning </td><td> Trin Brugt </td></tr><tr><td> Lyse / Vask </td><td> 50 mM NaH 2 PO4 300 mM NaCI 10 mM imidazol, pH 8,0 </td><td> 1,4 </td></tr><tr><td> Tris / salt </td><td> 50 mM Tris, pH 8,0 300 mM NaCI </td><td> 1,7 </td></tr><tr><td> Elueringspuffer </td><td> 50 mM Tris, pH 8,0 300 mM NaCI 250 mM imidazol </td><td> 1,8 </td></tr><tr><td> RBC Assaypuffer </td><td> 0,3% BSA 2 mM CaCl2 10 mM HEPES, pH 7,4 </td><td> 2,1 </td></tr><tr><td> puffer R / B </td><td> RPMI-cellekulturmedium 2 mM CaCl2 0,5% BSA </td><td&gt; 3,1 </td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> Tabel 2. Liste over buffere, der anvendes i denne protokol. De anvendte buffere, er deres sammensætning og det første skridt, hvor de anvendes i protokollen angivet. </td></tr></tbody></table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

Bakterielle toksiner binder til kolesterol i membraner, der danner porer, som tillader lækage af cellulære indhold og tilstrømning af materialer fra det ydre miljø. Cellen kan enten komme sig efter denne fornærmelse, som kræver aktive membran reparationsprocesser, ellers dør afhængigt af mængden af toksin eksponering og celletype 1. Endvidere inducerer disse toksiner kraftige inflammatoriske reaktioner i inficerede værter ved aktivering af immunceller, herunder makrofager, som producerer en række pro-inflammatoriske cytokiner 2. Mange Gram-positive bakterier producerer kolesterol bindende toksiner, der har vist sig at bidrage til deres virulens gennem stort set ukarakteriserede mekanismer. Morfologiske ændringer i plasmamembranen i celler udsat for disse toksiner indbefatter deres binding til cholesterol-berigede overfladefremspring, som kan udskilles til det ekstracellulære rum, hvilket antyder en indre cellulært forsvarmekanisme 3,4. Denne proces sker på alle celler i fravær af metabolisk aktivitet, og kan visualiseres ved anvendelse EM efter kemisk fiksering 4. I immunceller, såsom makrofager, som medierer inflammation som reaktion på toksin eksponering, der induceres membranvesikler foreslået at indeholde cytokiner af IL-1-familien og kan være ansvarlige for både kaste toksin og videregiver disse pro-inflammatoriske cytokiner 5,6,7. En forbindelse mellem IL-1β frigørelse og en særlig type af celledød, er betegnet pyroptosis blevet foreslået, som begge er caspase-1 afhængige processer 8. At sortere de mange aspekter af dette makrofag reaktion, som omfatter toxinbinding, kaste af membranvesikler, cytokinfrigivelse og potentielt celledød, har vi udviklet mærkningsteknikker og fluorescensmikroskopi metoder, der giver mulighed for real time visualisering af toksin-celle-interaktioner, herunder målinger af dysfunktion og død (figur 1). Brugaf levende celler er nødvendig på grund af begrænsninger i andre teknikker. Biokemiske indfaldsvinkler kan ikke løse effekter forekommer i individuelle celler, mens flowcytometri ikke tilbyder høj opløsning, real-time visualisering af de enkelte celler. Fremgangsmåderne beskrevet her kan anvendes til kinetisk analyse af responser induceret af andre stimuli medfører komplekse fænotypiske forandringer i cellerne.

Typisk 10 7 -10 8 U / ml SLO kan opnås med en proteinkoncentration på 4 mg / ml. Mængden af toksin kræves til cellelysis afhænger af celletypen, men er sædvanligvis fra 125 til 500 U / ml SLO (figur 2B). Celletyper som makrofager kan være mere resistente (4000 U / ml) selvom andre (især T-cellelinier) er mere følsomme. Disse følsomheder svarer til kommercielt tilgængelige SLO. Toksinaktivitet falder omtrent 2 gange med hver fryse-tø, så hemolytiske assays med hvert par...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Fremgangsmåder til oprensning af cholesterol binding toksin streptolysin O fra rekombinant<em&gt; E. coli</em&gt; Og visualisering af toxin binding til levende eukaryote celler er beskrevet. Lokal afgivelse af toksin inducerer hurtige og komplekse ændringer i målceller afslører hidtil ukendte aspekter af toksin biologi.

Visualisering af bakterielt toksin Inducerede Responses Brug af Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

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