| Overview | PrinterFriendlyVersion  | 
| Ex/Em(nm) | 652/None | 
| MW | N/A | 
| CAS# | N/A | 
| Solvent | N/A | 
| Storage | F/D/L | 
| Category | SmallMoleculeDetection MetalIons  | 
| Related | DiagnosticMolecules BiochemicalAssays  | 
AssayProtocol
1. Calciumstandardsandtestsamplepreparations:
1.1 Take10μLof300mMCalciumStandardSolution(ComponentC)to990µLDilutionBuffer(ComponentB)toget3
mMCalciumstandardsolution.
1.2 Add50μLof3mMCalciumStandardsolution(fromStep1.1)to950µLDilutionBuffer(ComponentB)toget150µM
Calciumstandardsolution.
1.3 Take500μLof150µMCalciumStandardSolution(fromStep1.2)toperform1:2serialdilutionstoget75,37.5,18.75,
9.375,4.68,2.34and0µMseriallydilutedCa2+standards.
1.4 Dilutethetestsampleto5-100µMrangewithDilutionBuffer(ComponentB).
1.5 Add50μLofCalciumStandard,dilutedsamplesandblankcontrolintoeachwellasshowninTable1.
Table1.Layoutofcalciumstandardsandtestsamplesinaclearbottom96-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.
Table2.Reagentcompositionforeachwell
CalciumStandard  | BlankControl  | TestSample  | 
Serialdilutions*(50μL)  | DilutionBuffer(50μL)  | 50μL  | 
*Note:Addtheseriallydilutedcalciumstandardsfrom150µMto2.34µMintowellsfromCS1toCS7induplicate.
2. Runcalciumassay:
2.1 Add50μLofCalciumBlue™(ComponentA)toeachwellofcalciumstandards,blankcontrol,andtestsamples
(seeStep1.5,Table1and2)tomakethetotalcalciumassayvolumeto100µL/well.
Note: Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
2.2 Incubatethereactionfor5-10minutesatroomtemperature,protectedfromlight.
2.3 Measuretheabsorbanceintensitiesat600nmor650nm.
3. Calciumcalibrationcurve:
Plottheassaydataasshownbelow.

Figure1.Calciumdoseresponsewasmeasuredona96-wellblackwall/clearbottomplatewiththeAmplite™Colorimetric
CalciumQuantitationKit.Aslowas~2.5µMCa2+wasdetectedwith5minutesincubationtime(n=3).
AssayProtocolforSerumorUrineSamples
1. Preparecalciumstandardsandtestsamplepreparations:
1.1 Take10μLof300mMCalciumStandardsolution(ComponentC)to990µLDilutionBuffer(ComponentB)toget3mMCalciumStandardSolution.
1.2 Take500μLof3mMCalciumStandardSolutiontoperform1:2serialdilutionstoget1.5,0.75,0.375,0.1875,0.094,0.047and0µMseriallydilutedCa2+standards.
1.3 Add10μLofcalciumstandard,serumorurinesamplesandblankcontrolintoeachwellasshowninTable3.
Table3.Layoutofcalciumstandardsandtestsamplesinaclearbottom96-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.
Table4.Reagentcompositionforeachwell

		