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Ex/Em(nm)	575/None
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	CellBIOLOGy CellMetabolism
Related	RedoxEnzymes BiochemicalAssays

Lacticacidischiralandhastwoopticalisomers:L-lacticacidandD-lacticacid.Lactateisconstantlyproducedfrompyruvateviatheenzymelactatedehydrogenase(LDH)intheprocessofmetabolismandexercise.Monitoringlactatelevelsisagoodwaytoevaluatethebalancebetweentissueoxygendemandandutilizationandisusefulwhenstudyingcellularandanimalphysiology.D-lactateisnotmetabolizedbymammalsanditseliminationfromthebodydependsmainlyonrenalexcretion.D-andL-lacticacidarefoundinmanyfermentedmilkproductssuchasyoghurtandcheese,andalsoinpickledvegetables,andcuredmeatsandfish.TheD-andL-lacticacid(generatedbybacteria)isaqualityindicatoroffoods,suchasegg,milk,fruitjuiceandwine.AbnormalhighconcentrationofD-lactateinthebloodisusuallyareflectionofbacterialovergrowthinthegastrointestinaltract.AATBioquest"sAmplite™LactateAssayKits(Cat#13814and13815forL-lactateassay,andCat#13810and13811forD-lactateassay)providebothfluorescenceandabsorbance-basedmethodfordetectingeitherL-lactateorD-lactateinbiologicalsamplessuchasserum,plasma,urine,aswellasincellculturesamples.Intheenzymecoupledassay,lactateisproportionallyrelatedtoNADH,whichisspecificallymonitoredbyachromogenicNADHsensor.Thesignalcaneasilyreadbyanabsorbancemicroplatereaderattheabsorbanceratioof~A575nm/A605nmtoincreaseassaysensitivity.WiththisColorimetricAmplite™D-LactateAssayKit,wewereabletodetectaslittleas4µMD-lactateina100µLreactionvolume.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareNADstocksolution(100X):

Add100µLofH₂OintothevialofNAD(ComponentC)tomake100XNADstocksolution.

 

2.PrepareD-lactatestocksolution:

Add200µLofH₂Oor1xPBSbufferintothevialofD-lactateStandard(ComponentD)tomake100mMD-lacatestandardsolution.

Note:TheunusedD-lacatestandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat

-20^oC.

 

3.PrepareserialdilutionsofD-lactatestandard(0to1mM):

3.1   Add10μLofD-lactatestocksolution(fromStep2)into990µL1xPBSbuffertogenerate1mMD-lactatestandardsolution.

Note:DilutedD-lactatestandardsolutionisunstable,andshouldbeusedwithin4hours.

 

3.2   Take200μLof1mMD-lactatestandardsolutiontoperform1:3serialdilutionstoget300,100,30,10,3,1and0μMserialdilutionsofD-lactatestandard.

 

3.3   AddserialdilutionsofD-lactatestandardandD-lactatecontainingtestsamplesintoawhiteclearbottom96-wellmicroplateasdescribedinTables1and2.

 

Table1LayoutofD-lactatestandardsandtestsamplesinawhiteclearbottom96-wellmicroplate

BL	BL	TS	TS	….	….
Lac1	Lac1	….	….	….	….
Lac2	Lac2
Lac3	Lac3
Lac4	Lac4
Lac5	Lac5
Lac6	Lac6
Lac7	Lac7

               Note:Lac=D-LactateStandards,BL=BlankControl,TS=TestSamples.

 

Table2Reagentcompositionforeachwell

D-LactateStandard	BlankControl	TestSample
SerialDilutions*:50μL	DilutionBuffer:50μL	50μL

*Note:AddtheseriallydilutedD-Lactatestandardsfrom1μMto1mMintowellsfromLac1toLac7induplicate.

 

4.PrepareD-lactateassaymixture:

4.1   Add5mLofAssayBuffer(ComponentB)intoonebottleofEnzymeMix(ComponentA).

 

4.2   Add50µLNADstocksolution(100X,fromStep1)intothebottleofComponentA (fromStep4.1), and

mixwell.

Note:ThisD-lactateassaymixtureisenoughforone96-wellplate.Itisunstable,andshouldbeusedpromptlywithin2hoursandavoidexposuretolight.

Note2:Alternatively,onecanmakea50XofD-LactateEnzymeMixturestocksolutionbyadding100μLofH2OintothebottleofComponentA,andthenpreparetheD-lactateassaymixturebymixingthestocksolutionwithassaybuffer(ComponentB)and100xNADsolutionproportionally.Aliquotandstoretheunused50XD-LactateEnzymeMixturestocksolutionand100XNADsolutionat-20^oC,andavoidfreeze-thawcycles.

 

5.RunD-lactateassay:

5.1   Add50μLofD-lactateassaymixture(fromStep4.2)toeachwellofD-lactatestandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalD-lactateassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofD-lactateassaymixtureintoeachwell.

 

5.2   Incubatethereactionatroomtemperaturefor30minutesto2hours,protectedfromlight.

5.3   MonitortheabsorbanceratioincreasewithanabsorbanceplatereaderatA_575nm/A_605nm.

References&Citations

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Journal:PharmacologicalResearch(2017)

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Journal:JournalofMedicinalChemistry(2017)

NeuronalCultureMicroenvironmentsDeterminePreferencesinBioenergeticPathwayUse
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Authors:XiaofangZhang,LeiWang,XiaodongZhang,LijunRen,WenjingShi,YijunTian,JiangboZhu,TianbaoZhang
Journal:FoodandChemicalToxicology(2017)

Highglucoseandinterleukin1β-inducedapoptosisinhumanumbilicalveinendothelialcellsinvolvesindown-regulationofmonocarboxylatetransporter4
Authors:DongWang,QingjieWang,GaoliangYan,YongQiao,LingSun,BoqianZhu,ChengchunTang,YuchunGu
Journal:Biochemicalandbiophysicalresearchcommunications(2015):607--614

IntracellularATPdecreasemediatesNLRP3inflammasomeactivationuponnigericinandcrystalstimulation
Authors:JohjiNomura,AlexanderSo,MizuhoTamura,NathalieBusso
Journal:TheJournalofImmunology(2015):5718--5724

地衣芽胞杆菌对实验性结肠炎相关肝脏病变的疗效及机制影响
Authors:
Journal:世界华人消化杂志(2015):4399--4405

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