| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 551/577 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection MetalIons |
| Related | DiagnosticMolecules BiochemicalAssays |
Note:Thawallthekitcomponentstoroomtemperaturebeforestartingtheexperiment.
1.Preparestocksolutions:
Prepare200XRhodRed™stocksolutionbyadding50µLofsterileH2OintothevialofRhodRed™Indicator(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionneedstobealiquotedandrefrozenat-20oC.
2.Prepareassayreactionmixture:
Prepareassayreactionmixtureaccordingtothefollowingtable,keptfromlight.
Table1.Assayreactionmixtureforone96-wellplate
Components | Volume |
RhodRed™stocksolution(200X,fromStep1) | 25μL |
AssayBuffer(ComponentB) | 5mL |
TotalVolume | 5.025mL |
Table2.Layoutofcalciumstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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CS1 | CS1 | …. | …. | …. | …. |
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CS2 | CS2 |
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CS3 | CS3 |
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CS4 | CS4 |
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CS5 | CS5 |
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CS6 | CS6 |
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CS7 | CS7 |
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Note:CS=CalciumStandards,BL=BlankControl,TS=TestSamples.
Table3.Reagentcompositionforeachwell
CalciumStandard | BlankControl | TestSample |
Serialdilutions*:50μL | H2O:50μL | 50μL |
*Note:Addtheseriallydilutedcalciumstandardsfrom3mMto0.003mMintowellsfromCS1toCS7induplicate.
3.RunCalciumassay:
3.1 Prepareacalciumstandardbydilutingtheappropriateamountofthe300mMCalciumStandard(ComponentC)intoH2OtoproduceaCalciumconcentrationrangingfrom0to3mM(12mg/dL).A0 mMcalciumcontrolisincludedasblankcontrol.Thefinalcalciumconcentrationswillbetwofoldslower(i.e.,0to1.5mM)withtheadditionofassayreactionmixture(SeeStep3.3).
3.2 Add50μLofserialdilutedcalciumstandard(fromStep3.1)intoeachwell.
3.3 Add50μLofassayreactionmixture(fromStep2,Table1)toeachwellofcalciumstandard,blankcontrol,andtestsamples(seeStep2,Table3)tomakethetotalcalciumassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
3.4 Incubatethereactionfor5to30minutesatroomtemperature,protectedfromlight.
3.5 MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=540/590nm.
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Highefficiencysingle-stepBiomaterial-basedmicroparticlefabricationviatemplate-directedsupramolecularcoordinationchemistry
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Journal:Toxicon(2016):45--55
