Staining Procedure for Flow Cytometric Detection of Human Cyclins相关交流-蚂蚁淘在线

Staining Procedure for Flow Cytometric Detection of Human Cyclins

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ThisisastandardprotocolusedatPharmingenforQualityControltestingoftheanti-cyclinantibodiesbyflowcytometry.Itisagoodstartingpoint.However,investigatorsmayneedtooptimizeprotocolsfortheirownexperimentalsystem.Itisparticularlyusefultorefertothepublishedliteratureregardingprotocolstypicallyusedforagiventypeofprotein.

MATERIALS:12x75mmtesttubes,PipetmanPipettes(P-20,P-200andP-1000),50mlconicaltubes,Pipettips,Tabletopcentrifugeorequivalent,PermanentMarker,Aspirator

BUFFERS:PhosphateBufferedSaline(PBS):(Cat.No.21428A;3bottlesof125mleach):140mMNaCl,2.7mMKCl,10mMNa₂HPO₄,1.8mMKH₂PO₄dissolvedindistilled,water.ThepHhasbeenadjustedto7.2usinghydrochloricacid.WashBuffer:PBS/0.1%NaN3/1%heat-inactivatedfetalbovineserum.ThepHoftheWashbuffershouldbe7.1–7.4.

REAGENTS:Washbufferstoredat4°C,75%ethanolstoredat–20°C,Puremethanolstoredat-20°C,1%formaldehyde(methanol-free)inPBS,storedat4°C,0.25%Triton®X-100inWashbuffer,storedat4°C,propidiumiodide(PI)solution:10µg/mlPIinPBS,storedat4°C.

Procedure:

Fixation

1.	Harvest,countandpelletcellsfollowingstandardprocedures.
2.	WashcellsoncebyresUSPendingthepelletin30-40mlofWashbuffer.Centrifugeat200xgfor10minandaspiratesupernatant.
3.	Whilevortexing,add10mlcold75%ethanol(forD1,usepurecoldmethanol),dropbydrop,tothecellpellet.
4.	Incubateat–20°Cforaminimumof2hr.Thefixedcellscanbestoredat–20°Cin75%ethanolforupto30days. Note:ForD-typecyclins(besidesD1)thecellsshouldfirstbefixedin5mlof1%methanol-freeformaldehydeinPBSfor15minonice(orat4°C)priortofixationwithethanol.Followingincubationwithformaldehyde,centrifugeat200xgfor10minandaspiratesupernatant.Resuspendpelletin30-40mlWashBuffer,centrifugeat200xgfor10minandaspiratesupernatant.ThengotoStepNo.3above.

II.

Staining

1.	Justpriortostaining,removeethanolbycentrifugationat200xgfor10min.Aspirateandwashoncebyresuspendingpelletin30-40mlofWashbuffer.Centrifugeat200xgfor10min.Aspiratesupernatant.
2.	Add5mlcold0.25%Triton®X-100inWashbuffertothecellpellet,vortexandincubate5minonice(orat4°C).
3.	Add30-40mlWashbuffertotheabovesuspensionandcentrifugeat200xgfor10min.Aspiratesupernatant.
4.	ResuspendpelletinWashbuffertoafinalconcentrationof1x10⁷cells/ml.
5.	Aliquot100mlcellsuspension(1x10⁶cells)into12x75mmtubesforstaining.
6.	Add20mlofanti-cyclinorisotypecontrolantibodyatoptimalworkingdilution.Incubate30minatRTinthedark.
7.	Add2mlWashbuffer,centrifugefor5minat200xg.Aspiratesupernatant.
8.	IfusingdirectlyconjugatedisotypecontrolsandmAbs,gotoStep10below.
9.	IfusingunconjugatedisotypecontrolsandmAbs,add20mlofFITC-conjugatedgoatanti-mouseIg(Cat.No.12064D)atoptimalworkingdilution.Incubate30minatRTinthedark.Add2mlWashbuffer,centrifuge5minat200xgAspiratesupernatant.
10.	Resuspendcellpelletin0.5mlPIsolutionforsimultaneousanalysisofDNAcellcycleandcyclinexpression.Incubatecellsat4°CinPIfor20minpriortoanalyzingbyflowcytometry.Analyzestainedcellswithin4hr.Storeat4°Cinthedarkpriortoanalysis.

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Staining&nbsp;Procedure&nbsp;for&nbsp;Flow&nbsp;Cytometric&nbsp;Detection&nbsp;of&nbsp;Human&nbsp;Cyclins

Staining Procedure for Flow Cytometric Detection of Human Cyclins