53$1-1 epvcmf tubjo,ju - takara bio

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

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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TRACP & ALP double-stain KitCat. #MK300

v202001Da

URL:http://www.takara-bio.com

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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(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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53$1-1 epvcmf tubjo,ju - takara bio

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