BrdU Staining Protocol
来自 : 蚂蚁淘
Supplies:Ethanol100USP(highestquality)FACSStainingBuffer(1XPBSw/3calfserum,0.05azide--filtered)—DilutestainingantibodiesinBufferDNAse(SigmaD-5025,BovinePancreas)RNase(Boehringer,25mgbovinepancrease)Anti-BrdU-FITC(BectonDickinsonorPhoenixFlow)0.15MNaCl,1.5MNaCl10Paraformaldehype(keptasstockin-80°C)Tween1MMgCl2FACSTubes
Protocol:
Cellsin96wellFACSplate
- Blockwith24G-2
- Surfacestaincellsasusual-Omitfourthchannellabeledantibodiesonallstains;EtOHdestroysAPC
- PreparetubesfromwhichtotransferEtOHdropwise(1.2mlEtOHonICE)
- Resuspendcellsfrom96wellplacewith100µl0.15MNaCl(cold)
- TransfertoFACStubesONICE.Add400µl0.15MNaCltoeachtube
- Vortexat1/3speedandaddEtOHwithpasteurPipetteat1droppersecond.Thisisacriticalstep...donotaddEtOHtooquickly
- Incubateonicefor30minutes
- Spin10minutes@2000RPM,4°C
- Dumpandshakeliquidintowaste
- Usingrepeatpipetter,squirt1mlFACSstainingbufferintoeachtube
- Spin10minutesanddumpasbefore(step8)
- Add1ml1paraformaldehyde+0.05Tween10-For20ml:2.0ml10paraformaldehyde10µlTween-20
- Incubateatroomtemperaturefor30minutes
- Incubateonicefor30minutes
- Spinanddumpasbefore(step8)Add1mlDNAse(0.15MNaCl+4.2mMMgCl+100Kunitzunits/mlDNAse)-For50ml:
46.5mLdH20200ulMgCl2(1Mstock)1500uLNaCl(5Mstock)100KunitzunitsDnase(volumedependsonactivityofbatch)Incubatefor30minutes@25°
- Spin10min.anddumpasbefore(step8)
- TransfercellsfromFACStubesto96-wellplate.Washoncewithstainingmedia.
- Blockwith10ratserum.Incubate15minuteonice.Spinanddumpasbefore(step8:itiscriticaltospinathighspeedoncethecellshavebeenfixedwithEtoH/PFAsincetheybecomelessdense).
- Addanti-BrdU-FITCorbiotin(1:20dilutionforPhoenixflow).
- Pipetteupanddowntoresuspendpellet.Incubatefor30minutesonice(orovernightat4°C).
- Washanddumpasbefore.TransfercellsintoFACStubes.
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