Name: a fluorescent screening assay for identifying modulators of girk channels
Uploaded: 2012-04-24T13:00:00
Description: Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

Dette arbeidet ble støttet av US Public Health Service Award NS-071530.

1. Utarbeidelse av celler <ol><li> Grow hypofysen AtT20 celler i Dulbecco modifiserte Eagle medium (DMEM) supplert med 10% hest serum og vedlikeholde kulturer ved 37 ° C i en fuktet atmosfære av 5% CO 2. </li><li> Opprettholde kulturen ved subculturing cellene hver 5 til 7 dager ved hjelp av en standard trypsinization prosedyre. </li><li> Smør brønner av svart, klar bunn, 96-og tallerkener med 50 mL av poly-L-lysin. La brønnene tørke i 30 min i kuvøse. </li><li> Plate cellene i 96-brønners plater so...

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Mens membranpotensiale-sensitive fluorescerende fargestoffer blitt brukt til å identifisere stoffer som modulerer ionekanaler 9,10, er dette den første rapporten om deres søknad om nevronale GIRK kanal legemiddelforskning. Den GIRK kanalen fluorescerende analysen presenteres her gir en rask, pålitelig og real-time metode for screening av ligand-gated K +-kanaler. Analysen kan modifiseres for bruk med et bredt spekter av celler, inkludert udødeliggjorde celler linjer (HEK293, CHO, etc.) som utt...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> DMEM </td><td> CellGro </td><td> 10-013 </td></tr><tr><td> Horse serum </td><td> Invitrogen </td><td> 16050-114 </td></tr><tr><td> 96 brønner plater </td><td> Corning </td><td> 3603 </td></tr><tr><td> Poly-L-lysin </td><td> Sigma-Aldrich </td><td> P4707 </td></tr><tr><td> Somatostatin </td><td> Sigma-Aldrich </td><td> S9129 </td></tr><tr><td> Carbachol </td><td> Sigma-Aldrich </td><td> C4382 </td></tr><tr><td> HLB 021-152 </td><td> AnaSpec </td><td> 89300 </td></tr><tr><td> Versette automatisert væske behandleren </td><td> ThermoFisher </td><td> 650-01 </td></tr><tr><td> Synergy2 fluorescerende plateavleseren </td><td> BioTek </td><td></td></tr><tr><td> Gen5 analyse software </td><td> BioTek </td><td></td></tr></tbody></table> Tabell 1. Tabell av spesifikke reagenser og utstyr.

a fluorescent screening assay for identifying modulators of girk channels

G protein-gated innover likeretter K + (GIRK) kanaler fungere som cellulære mediatorer av et bredt spekter av hormoner og signalstoffer og er uttrykt i hjernen, hjertet, skjelettmuskulatur og endokrine vev 1,2. GIRK kanaler blir aktivert etter binding av ligander (nevrotransmittere, hormoner, medikamenter osv.) til sin plasma membran-bundet, G protein-koblede reseptorer (GPCRs). Denne bindingen fører til stimulering av G-proteiner (G jeg og G o) som senere binde seg til og aktivere GIRK kanalen. Når åpnet GIRK kanalen tillater bevegelse av K + ut av cellen som forårsaker hviler membran potensial til å bli mer negativ. Som en konsekvens, minsker GIRK kanal aktivering i nevroner spontan handling potensial formasjon og hemmer frigjøring av eksitatoriske nevrotransmittere. I hjertet, hemmer aktivering av GIRK kanalen pacemakeren aktivitet og dermed bremse pulsen. <pclass = &quot;jove_content&quot;&gt; GIRK kanaler representere nye mål for utvikling av nye terapeutiske agenter for behandling nevropatisk smerte, narkotikaavhengighet, hjertearytmier og andre lidelser tre. Imidlertid fortsatt farmakologi av disse kanalene i stor grad uutforsket. Selv om en rekke medikamenter, inkludert antiarytmiske midler, antipsykotika og antidepressiva blokkere GIRK kanal, er dette hemming ikke selektiv og forekommer ved relativt høye konsentrasjoner narkotika 3. Her beskriver vi en real-time screening test for å identifisere nye modulatorer av GIRK kanaler. I denne analysen, er nevrale AtT20 celler, uttrykker GIRK kanaler, lastet med membranpotensiale-sensitive fluorescerende fargestoffer som bis-(1,3-dibutylbarbituric syre) trimethine oxonol [DiBAC 4 (3)] eller HLB 021-152 (Figur 1 ). De fargestoffer blitt sterkt fluoriserende følgende opptak i cellene (Figur 1). Behandlingav cellene med GPCR ligander stimulerer GIRK kanaler for å åpne. Den resulterende K + effluks ut av cellen fører til at membranen potensial til å bli mer negativ og fluorescerende signal å redusere (figur 1). Dermed kan medikamenter som modulerer K + efflux gjennom GIRK kanalen bli analysert ved hjelp av en fluorescerende plateavleseren. I motsetning til andre ionekanal screening-analyser, slik atomabsorpsjonspektrometri 4 eller radiotracer analyse 5, gir GIRK kanalen fluorescerende analysen en rask, real-time og billig screening prosedyre.

Watch this Scientific Journal Video about A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels at JoVE.com

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

En sanntid screening prosedyre for å identifisere stoffer som samhandler med G protein-gated innover likeretter K<sup&gt; +</sup&gt; (GIRK) kanaler er beskrevet. Analysen benytter membran potensial-sensitive fluorescerende fargestoffer for å måle GIRK kanal aktivitet. Denne teknikken er tilpasningsdyktig for bruk på en rekke cellelinjer.

En fluoriserende Screening Assay for å identifisere modulatorer av GIRK kanaler

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

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