外周血和脐带血中分离单个核细胞—FicollHypaque梯度离心法...
来自 : 蚂蚁淘
StewartLaboratoryStandardOperatingProcedure
BrdUIncorporationProtocol
Day1
- Labelcellswithmediacontaining100μMBrdU(6μL/mLof5mg/mLstock):
- DiploidFibroblasts,4-6hours
- EScells,30minutes
- 3T3cells,2.5hours
- Trypsinizecells,washfromplatewithPBS,spindown(400xg,1700rpm,5minin15mltubes-nofaster!),andresUSPendin10mL70%coldEtOH.
- Storeat-20°Covernightorforseveraldays
Day2
- Pelletcellsandwashoncewithwashbuffer(PBS+0.5%IFS).
- Pelletandresuspendin0.5mL2MHCl+0.5%IFS(makefresh!).IncubateatRTfor20minutes.
- Washcellsoncewith1mLwashbuffer.
- Pelletandresuspendin0.1Msodiumtetraborate(Na2B4O7).IncubateatRTfor2minutes.
- Washcellstwicewith1mLwashbuffer(ifanegativecontrolofPIalonestainisdesired,setasideafractionofcellsuntilstep10).
- Resuspendcellpelletinanti-BrdUantibody(50μLtotalsolutiondilutedwithwashbuffer).Incubatefor20minutesat4°C.
UseBrdU-FITC:Boeringher#12026931:100dilution(skiptostep9)
Oranti-BrdU:Boeringher#11703761:10dilution
- Washcellsoncewith1.5mLwashbuffer.
- Resuspendcellpelletinanti-mouseFITCantibody(50μLtotalsolutionwithwashbuffer,1:500dilution).Incubatefor20minutesat4°C.
- Washcellsoncewith1.5mLwashbuffer.
- Resuspendpelletin0.3mLPI(10μg/mLinwashbuffer).Incubatefor30minutesatRT.
- Cellsarereadyforanalysisbyflowcytometry.
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