Overview

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Ex/Em(nm)	None/None
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	N2(L)
Category	GPCR cAMPGPCRAssays
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ScreenQuest™celllinesareaseriesofcellsthathavebeensuccessfullyusedindrugdiscoveryandscreeningenvironmentsforstudyingG-protein-coupledreceptors(GPCR)thatdonotconventionallycouplethroughintracellularcalcium.IthavebeeneffectivelyusedwiththeFLIPR,FDSSSystemsinconjunctionwithnon-Gqcoupledmembersofmanyreceptorssuchaschemokine,SEROtonin,glutamate,dopamine,opoid,vasopressinand?-andß-adrenoceptorreceptorfamilies.Over60%oftheknownG-protein-coupledreceptorssignalthroughpathwaysotherthanGqwhichleadtoanincreaseinintracellularcalcium,andasgenomicsrevealsmoreG-protein-coupledreceptortargetsthistrendcontinuestoincrease.ScreenQuest™celllinesareusedforinvestigatingG-protein-coupledreceptors(GPCR)thatdonotconventionallycouplethroughintracellularcalcium.ScreenQuest™celllinesarebasedonaseriesofG-proteinchimeras,includingthepromiscuousG-protein,G?16.ThechimerasconsistofthealphasubunitofaGq-proteincomplexwhose5carboxy-terminalaminoacidshavebeenreplacedwiththosefromoneoftheotherG-proteins(eitherG?s,G?i,G?o,orG?z).TheseaminoacidsareresponsIBLeforthecouplingofthereceptortoitsG-protein.Co-expressionofthesechimeraswithspecificnon-Gq-coupledreceptorswhichnormallyactthroughthecAMPpathwaymayresultinthegenerationofanintracellularcalciumsignaluponreceptorstimulation.ScreenQuest™CHO-GqocelllineisCHO-K1cellsstablytransfectedwiththechimericGqoalphasubunitprotein.WhenusedasahostcellfortransfectionexpressionofGi-coupledreceptors,theconstitutivelyexpressedGqoproteininthecellsallowsthetransfectedreceptorwhichnormallyactthroughthecAMPpathway,tocoupletoGqsignaltransductionandmobilizedintracellularcalcium.Activationofthespecificnon-Gq-coupledreceptorsinthesecellsbyspecificligandscanbedetectedusingcalciumsensitivedyessuchasCal-520AM,Fluo-8AM,orFluo-4AM.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1. CellDensityandStorage
   Thecellsarefrozenatadensityof2X106cellsin90%fetalbovineserumand10%(v/v)DMSO.ToinsurethehighestlevelofviABIlity,thawthevialandinitiatethecultureassoonaspossibleuponreceipt.Cellsmustbestoredinliquidnitrogenifnotimmediatelyprocesseduponreceipt.
2. CellCultureMedium
   Basalmedium:Ham’sF12culturemediumcontaining10%fetalbovineserum,2mML-glutamine,100units/mLpenicillin,100µg/mLstreptomycin(Basalmedium).
   SelectionMedium:BasalMediumwith200µg/mLhygromycinBand400ug/mLG418.
3. ThawingandSeedingCells
   1. PrepareBasalMedium.Prepare37°CWaterBath.Thawcellsrapidlybyremovingfromliquidnitrogen,andimmediatelyimmersingina37°Cwaterbath.Removethevialfromthewaterbathassoonasthecontentsarethawed,andsterilizetheexteriorofthevialwith70%ethanol.Alloftheoperationsfromthispointonshouldbecarriedoutunderstrictasepticconditions.
   2. Addvialcontentsto25mLcellculturemediuminT75TissueCultureTreatedFlask.Gentlyswirlflaskandplaceinahumidified,tissuecultureincubator,37°C,5%CO2.
   3. Alllivecellsshouldbeattachedafter18-24hourspost–thaw.ChangeBasalMediumwithSelectionMedium.
4. SubcultureandPropagation
   1. Whencellsareapproximately80%confluent,passagethecells.
   2. Passagethecells1:10every3-4days,usingTrypsin-EDTAtodissociatethecells.
   3. Itishighlyrecommendedthatafrozencellbankbeestablishedatlowpassagenumber.

References&Citations

CitationExplorer

ActivationofP2X7andP2Y11purinergicreceptorsinhibitsmigrationandnormalizestumor-derivedendothelialcellsviacAMPsignaling
Authors:DAvanzato,TGenova,AFiorioPla,MBernardini,SBianco,BBussolati,DMancardi,EGiraudo,FMaione,PCassoni
Journal:ScientificReports(2016)

TheM2muscarinicreceptorsareessentialforsignalingintheheartleftventricleduringrestraintstressinmice
Authors:HanaTomankova,PaulinaValuskova,EvaVarejkova,JanaRotkova,JanBenes,JaromirMyslivecek
Journal:Stress(2015)

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