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From lot #AJG0826A onward, the kit contains "Fixation base solution", whose composition is Citrate buffer (pH 5.4), instead of "Fixation solution". Please add acetone and ethanol in the Fixation base solution before use. There is no change to the protocol and performance of the kit.
Cat. # MK300
Product Manual
TRACP & ALPdouble-stain Kit
For Research Use
v202001Da
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Table of Contents
I. Description........................................................................................................ 3
II. Introduction..................................................................................................... 3
III. Principles........................................................................................................... 4
IV. Components.................................................................................................... 5
V. Storage............................................................................................................... 5
VI. PreparationofReagents.............................................................................. 6
VII. Methods
Cellfixation.................................................................................................. 7
Activitystaining......................................................................................... 7
Nuclearstaining......................................................................................... 8
VIII. ApplicationExamples................................................................................... 9
IX. References.......................................................................................................13
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I.Description
Thisproductisastainingkitforbone-relatedcells.Chromogenicsubstratesforalkalinephosphatase,anenzymemarkerofosteoblasts,andtartrate-resistantacidphosphatase,anenzymemarkerofosteoclasts,arecombinedwithareagentfornuclearstainingthatprovidesvisualizationofmultinucleatedosteoclasts.Bothacidandalkalinephosphataseactivitiesinthecellscanbestainedsimultaneouslyforcomparison.Moreover,asthesubstratesareprovidedaspremixedreagents,thesubstratesolutionscanbeeasilyprepared.
II.Introduction
Phosphatasesareenzymesthatactonaliphaticandaromaticphosphateestersandhydrolyzethemtoreleasephosphates.AlkalineandacidphosphataseshaveoptimumpHsforactivityatalkalineandacidpHs,respectively.Acidphosphatasesarepresentinavarietyofcellsandtissues,suchasprostate,liver,kidney,spleen,erythrocyte,plateletandosteoclasts.1,2In1959,Burstonereportedthatpotentacidphosphataseactivityisfoundinosteoclastsandalkalinephosphataseactivityisfoundinosteoblasts.3Followingthisreport,variousstudieshaveshownphosphataseactivityassociatedwithosteocytes;theacidphosphataseactivityofosteoclastswasshowntoberetainedinthepresenceoftartrate(tartrate-resistantacidphosphatase,TRACPorTRAP).TRACPactivityisrequiredforproperosteoclastfunction.Inadditiontoosteoclasts,hairycellsamongbloodcellsarealsoknowntohaveTRACPactivity.Acidphosphatasesthatareinactivatedinthepresenceoftartratearetartrate-sensitiveacidphosphatases(TSACPorTSAP).Alkalinephosphatasesaremembrane-boundglycoproteinsthatareclassifiedintofourtypes:intestinal,placental,placenta-like,andtissuenon-specific.Amongthetissuenon-specifictypealkalinephosphatases,thebone-specificisozymeiscalledbonetypealkalinephosphatase.Thisenzymeisboundtothemembraneofosteoblastsandfunctionstoenhanceosteogenesisbydegradingpyrophosphatethatinhibitscrystallizationatthecalcificationsiteandbydegradingorganicphosphateesterstoincreasetheinorganicphosphateconcentration.Giventhisfunction,bonetypealkalinephosphataseisusedasamarkerofosteogenesisduringbonemetabolism.Sincebonemetabolisminvolvesmutuallybalancedosteogenesisandboneresorption,simultaneousestimationwithtwoenzymemakersisuseful.
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TRACP & ALP double-stain KitCat. #MK300
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(2) PrincipleforstainingofalkalinephosphataseThesubstratesolutionforalkalinephosphatase(BCIP/NBT)isaddedtocellsamplesfixedonmicroplatewellsorslideglasses.Asshownbelow,formazandyewithabluish-purplecolorisgeneratedinthepresenceofalkalinephosphatasethroughareactionmediatedbycomponentsinthesubstratesolution.
AcidphosphataseNaphthol-AS-BI-phosphate HPO42- + naphthol-AS
FastRedVioletLB(diazoniumsalt)Azo dye(purplishred);pH 5.2
Alkalinephosphatase
NitroBlueTetrazoliumChloride
Bromo-Chloro-Indolyl phosphate HPO42- + Br-Cl-Indol
Formazan dye(bluishpurple);pH 9.5
III.Principles
(1) Principleforstainingofacidphosphatases(tartrate-resistantand-sensitiveacidphosphatases)1,2Forstaining,cellsfixedonmicroplatewellsorslideglassesareusedasthesample.Twosamplesfromthesameoriginareprepared,andthereactionisperformedbyaddingthesubstratesolutionforacidphosphatase(NABP/FRVLB)supplementedwithtartratetooneofthesamples,andthesubstratesolutionwithouttartrateisaddedtotheothersample.Thetartrate-resistantacidphosphatase(TRACP)activitycanbedetectedinthefirstsampleandthetotalacidphosphataseactivityincludingtartrate-resistantand-sensitivephosphataseactivitiescanbedetectedinthesecond.Asshownbelow,azoicdyewithapurplish-redcolorisgeneratedineachsampleinthepresenceoftheenzyme.Thisbyproductisdetectedbyreactionmediatedbycomponentscontainedinthesubstratesolution.
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IV.Components
(1)Fixationbasesolution 13.5mlCitratebuffer(pH5.4)Note:Acetonandethanolshouldbeaddedbeforeuse.
(2)Sodiumtartrate 4ml0.5Msodiumtartratebuffer(pH5.2)
(3)SubstrateforACP(premixed) for10mlx3NABP/FRVLB
(4)SubstrateforALP(premixed) for10mlx3BCIP/NBT
(5)Nuclearstain 10mlMethylgreen(Containsaceticacidinthesolvent.)
MaterialsRequiredbutNotProvided
・acetone(reagentgrade) 13.5ml・ethanol(reagentgrade) 2.7ml
Note: Addbothreagentto(1)Fixationbasesolution.RefertoVI.PreparationofReagents.
V.Storage
-20℃Eachreagentshouldbestoredatasuitabletemperatureafterfirstuse.
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VI.Preparation of ReagentsThiskitcontainsfor10mlx3ofpremixedsubstrateforeachenzyme.Thetotalamountofeachsubstratesolutionissufficientforstainingapproximatelyfive24-wellcultureplates.Whenperformingdouble-staining,detectionofacidphosphataseactivitymustprecedethestainingofalkalinephosphatase.StainingofalkalinephosphataseshouldbeperformedbyreplacingthesubstrateforALPafterdetectionofacidphosphatase.Notethatacidphosphatasewillbepartiallyinactivatedifthestainingiscarriedoutintheinverseorder.
(1) FixationbasesolutionThawatroomtemperaturebeforeuseandgentlyadd13.5mlofacetoneand2.7mlofethanoltoprepareFixationsolution.Acetoneandethanolofageneralreagentgradecanbeused.
* Useapipetteresistanttoorganicsolvents(e.g.,glasspipette)foracetoneaddition.
* Becarefultoaddacetoneandethanolbecausethesolutionreachesupperlimitofthecontainer.
* HandlewithcareasorganicsolventcontainedintheFixationsolutionisinflammable.
* TheFixationsolutioncanbestoredat-20℃afterpreparation,asitwillnotfreezeevenwhenstoredatorbelow-20℃.
* Evenifinsolublematerialsmaybeappearedduringstorage,thematerialswillbedissolvedatroomtemperature.
(2) SodiumtartrateThissolutionshouldbethawedatroomtemperaturepriortouse.
(3) SubstrateforACP(premixed)Thematerialinthevialshouldbedissolvedin10mlofsteriledistilledwaterwhenusedasthesubstratesolutionforthereactionofacidphosphatase.Thepreparedsolutionwillhaveaslightlyyellowcolor.Fordetectionofthetartrate-resistantenzyme,0.1volumeofSodiumtartrate(2)shouldbeaddedtothissolution.NABP/FRVLBandanoptimizedbufferarecontainedinthisproduct.Beforesubstratesolutionpreparation,storefrozenatorbelow-20℃.Thepreparedsubstratesolutioncanbestoreduptoonemonth.Thefrozensolutionmayformasmallamountofprecipitate.Insuchacase,thesolutionshouldbefilteredthrougha0.22-μmmembranebeforeuse.Thesubstratesolutioncontainingtartratealsocanbestoredinfrozen.
(4) SubstrateforALP(premixed)Onetabletofthiscomponentshouldbedissolvedwellin10mlofsteriledistilledwatertobeusedasthesubstratesolutionforthereactionofalkalinephosphatase.Preparationofthissolutionshouldbestartedatleast20minutesbeforeuse.Thepreparedsolutionwillhaveaslightlyyellowcolor.BCIP/NBTandanoptimizedbufferarecontainedinthisproduct.Beforesubstratesolutionpreparation,storefrozenatorbelow-20℃.Thepreparedsubstratesolutioncanbestoreduptoonemonth.Thefrozensolutionmaycontainasmallamountofprecipitate.Insuchacase,thesolutionshouldbefilteredthrougha0.22-μmmembranebeforeuse.
(5) NuclearstainThisreagentshouldbeuseddirectlyafterthawingatroomtemperature.Thethawedreagentshouldbestoredatorbelow20℃.Thisreagentcanbeusedforgeneralnuclearstainingorforexaminingwhetherosteoclastsaredifferentiatedandfusedintomultinucleatedcells.
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VII.Methods< Cell fixation >
A. Fixationofcellculturedin24-wellplates(exampleprotocolforfixationofbonemarrowcells)
1. Culturecellsina24-wellplate.2. RemoveanddiscardtheculturesupernatantandwashoncewithsterilePBS.3. Add200μlofFixationsolutionpreparedatStepVItoeachwell,placetheplateatroomtemperaturefor5minutes,andallowthecellstofixinthewell.
4. Add2mlofsteriledistilledwatertoeachwelltodilutethefixationsolution,andthenaspiratethesolution.Add2mlofsteriledistilledwateragaintowashthewell,andremoveanddiscardalltheliquidfromthewell.Samplescanbedriedafterthisstepandstoredatorbelow-20℃foratleastoneweek.
*TheamountoftheFixationsolutionissufficientforfixationoffive24-wellcultureplates.
B. Fixationofcellculturedin96-wellplatesFollowtheprocedureusedfor24-wellplatesbutuse50μlofFixationsolutionand250μlofsteriledistilledwaterforwashing.
*TheamountoftheFixationsolutionissufficientforfixationoffive96-wellcultureplates.
< Activity staining >
A. Singlestaining
1. PreparethesubstratesolutionforacidphosphataseoralkalinephosphataseaccordingtotheinstructionsinsectionVI[PreparationofReagents].Fordetectionoftartrate-resistantenzyme,add0.1volumeofSodiumtartrate(2)tothesubstratesolutionforacidphosphatase.
2. Addthesubstratesolutiontothewellorslideglassonwhichthecellsarefixed.CovertheplatewiththelidortheslidewithParafilmtopreventthesamplefromdrying.Amountofsubstratesolutiontobeused:
24-wellplate 200μl/well96-wellplate 50μl/wellSlideglass adequateamount
3. Incubateat37℃for15-45minutes.
Note: Theperiodforcolorformationwillvarydependingontheamountofphosphatasepresentinthecell.
4. Removeanddiscardthesolution,andwashthreetimeswithsteriledistilledwatertostopthereaction.
5. Examinethesamplebymicroscopy.(Steriledistilledwatercanbeaddedformicroscopicexamination.)
Note: Forstorageofstainedsamples,glycerolorthelikeshouldbeaddedtopreventdehydration.
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B. DoublestainingNote: Whenperformingdouble-staining,detectionofacidphosphataseactivity
mustprecedethestainingofalkalinephosphatase.StainingofalkalinephosphataseshouldbeperformedbyreplacingwiththesubstrateforALPafterdetectionofacidphosphatase.Notethatacidphosphatasewillbepartiallyinactivatedifthestainingiscarriedoutintheinverseorder.
1. Preparethesubstratesolutionforacidphosphataseandperformthereactionaccordingtotheprocedures1-3describedabove[A.Singlestaining].
2. Removeanddiscardthereactionsolutionandwashthreetimeswithsteriledistilledwater.
3. Preparethesubstratesolutionforalkalinephosphataseandperformthereactionaccordingtotheprocedures1-3describedabove[A.Singlestaining].
4. Removeanddiscardthereactionsolutionandwashthreetimeswithsteriledistilledwater.
5. Examinethesamplebymicroscopy.(Steriledistilledwatercanbeaddedformicroscopicexamination.)
Note: Forstorageofstainedsamples,glycerolorthelikeshouldbeaddedtopreventdehydration.
< Nuclear staining >Note: Stainingwithmethylgreenmayhinderthevisualizationofactivitystaining.
Examinebymicroscopyandconfirmtheresultsofactivitystainingpriortonuclearstainingwithmethylgreen.
1. Overlaytheactivity-stainedwellorslideglasswithNuclearstain(5).2. Incubateatroomtemperaturefor5minutes.3. Washwithsteriledistilledwater,andexaminebymicroscopyafteraddingglycerolortheliketopreventdehydration.
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Figure2.ActivitystainingofALPinculturedratbonemarrowcells.
VIII.Application Examples ・ Example 1
Bonemarrowcellscollectedfroma16-weekoldJWrabbit(male)wereculturedinthepresenceofM-CSFandactivevitaminD3.Cellswerestainedfortartrate-resistantacidphosphatase(TRACP)activityonday6oftheculture(Figure1).
Figure1.ActivitystainingofTRACPinculturedrabbitbonemarrowcells.
・ Example 2Bonemarrowcellscollected from24-dayoldSD rats (female)wereculturedinthepresenceofM-CSFandactivevitaminD3.Cellswerestainedforalkalinephosphatase(ALP)activityonday10ofculture(Figure2).
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・ Example 3Humanbonemarrowmononuclearcells(BIOWHITTAKER,INC.)wereculturedinthepresenceofdifferentadditivesubstances.CellswerestainedforTRACPandALPactivityseparatelywhenthecellsweredifferentiated(day9ofculture)(Figure3).
ActivitystainingofTRACP ActivitystainingofALP
+vitaminD3
+M-CSF
+vitaminD3 + M-CSF
+β-glycerophosphate+dexamethasone
Figure3.ActivitystainingofTRACPandALP.
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・ Example 4RatbonemarrowcellswereculturedinthepresenceofM-CSFandactivevitaminD3,anddifferentiated.DoublestainingofTRACPandALPactivitywascarriedoutonday12ofculture(Figure4).
Figure4.DoublestainingofTRACPandALPactivity.
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・ Example 5TheactivitystainingofTRACPandALPwascarriedoutseparatelyusingnewbornmouse(1day)frozentissuesections.250μlofFixationsolutionwasaddedto2frozenslides,andtheslideswereplacedatroomtemperaturefor5minutesforfixation.Slideswerewashedwellwithsteriledistilledwaterandextrawaterwasremovedwithapapertowel.250μlofsubstrateforAPwasaddedononeslideand250μlofsubstrateforTRACPwasaddedontheotherslide.Theslideswereincubatedat37℃for45minutes.Thesubstratesolutionwasremoved.Theslideswerewashedwithsteriledistilledwaterandextrawaterwasremoved.Slideswereobservedwithamicroscope.Theresultsusingfrozenslidesafterstoragefor6monthsat-80℃wasalmostsameastheresultsusingfreshfrozenslides(Figure5).
Figure5.ActivitystainingofALP&TRACPstaininginthenewbornmouse(1day)frozentissue.
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IX.References1)BurstoneMSetal .JNatlCancerInst .(1958)20:601-615.2)BurstoneMSetal .JNatlCancerInst .(1958)21:523-539.3)BurstoneMS.JHistochemCytochem.(1959)7:39-41.4)HarlowandLane.Antibodies,ALABORATORYMANUAL.(1988):406-407.
NOTE : Thisproductisforresearchuseonly.Itisnotintendedforuseintherapeuticordiagnosticproceduresforhumansoranimals.Also,donotusethisproductasfood,cosmetic,orhouseholditem,etc.Takaraproductsmaynotberesoldortransferred,modifiedforresaleortransfer,orusedtomanufacturecommercialproductswithoutwrittenapprovalfromTakaraBioInc.Ifyourequirelicensesforotheruse,pleasecontactusbyphoneat+81775656972orfromourwebsiteatwww.takara-bio.com.Youruseofthisproductisalsosubjecttocompliancewithanyapplicablelicensingrequirementsdescribedontheproductwebpage.Itisyourresponsibilitytoreview,understandandadheretoanyrestrictionsimposedbysuchstatements.Alltrademarksarethepropertyoftheirrespectiveowners.Certaintrademarksmaynotberegisteredinalljurisdictions.
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