53$1-1 epvcmf tubjo,ju - takara bio
TRANSCRIPT
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
2
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.
3
TRACP & ALP double-stain KitCat. #MK300
v202001Da
URL:http://www.takara-bio.com
(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.
4
TRACP & ALP double-stain KitCat. #MK300
v202001Da
URL:http://www.takara-bio.com
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.
5
TRACP & ALP double-stain KitCat. #MK300
v202001Da
URL:http://www.takara-bio.com
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.
6
TRACP & ALP double-stain KitCat. #MK300
v202001Da
URL:http://www.takara-bio.com
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.
7
TRACP & ALP double-stain KitCat. #MK300
v202001Da
URL:http://www.takara-bio.com
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.
8
TRACP & ALP double-stain KitCat. #MK300
v202001Da
URL:http://www.takara-bio.com
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).
9
TRACP & ALP double-stain KitCat. #MK300
v202001Da
URL:http://www.takara-bio.com
・ Example 3Humanbonemarrowmononuclearcells(BIOWHITTAKER,INC.)wereculturedinthepresenceofdifferentadditivesubstances.CellswerestainedforTRACPandALPactivityseparatelywhenthecellsweredifferentiated(day9ofculture)(Figure3).
ActivitystainingofTRACP ActivitystainingofALP
+vitaminD3
+M-CSF
+vitaminD3 + M-CSF
+β-glycerophosphate+dexamethasone
Figure3.ActivitystainingofTRACPandALP.
10
TRACP & ALP double-stain KitCat. #MK300
v202001Da
URL:http://www.takara-bio.com
・ Example 4RatbonemarrowcellswereculturedinthepresenceofM-CSFandactivevitaminD3,anddifferentiated.DoublestainingofTRACPandALPactivitywascarriedoutonday12ofculture(Figure4).
Figure4.DoublestainingofTRACPandALPactivity.
11
TRACP & ALP double-stain KitCat. #MK300
v202001Da
URL:http://www.takara-bio.com
・ Example 5TheactivitystainingofTRACPandALPwascarriedoutseparatelyusingnewbornmouse(1day)frozentissuesections.250μlofFixationsolutionwasaddedto2frozenslides,andtheslideswereplacedatroomtemperaturefor5minutesforfixation.Slideswerewashedwellwithsteriledistilledwaterandextrawaterwasremovedwithapapertowel.250μlofsubstrateforAPwasaddedononeslideand250μlofsubstrateforTRACPwasaddedontheotherslide.Theslideswereincubatedat37℃for45minutes.Thesubstratesolutionwasremoved.Theslideswerewashedwithsteriledistilledwaterandextrawaterwasremoved.Slideswereobservedwithamicroscope.Theresultsusingfrozenslidesafterstoragefor6monthsat-80℃wasalmostsameastheresultsusingfreshfrozenslides(Figure5).
Figure5.ActivitystainingofALP&TRACPstaininginthenewbornmouse(1day)frozentissue.
12
TRACP & ALP double-stain KitCat. #MK300
v202001Da
URL:http://www.takara-bio.com
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.
13
TRACP & ALP double-stain KitCat. #MK300
v202001Da
URL:http://www.takara-bio.com