DNA Cell Cycle
来自 : 蚂蚁淘
Solutions
- 70%ethanol
- ribonuclease(100µg/mlDNasefree,Sigma)
- propidiumiodide(50µg/mlinPBS)
Procedure
- Harvestcells.Spinat1200rpmfor5minutes.
- Discardsupernatant;add1ml"fridgecold"70%ethanolto1x106cellswhilevortexingthecellpelletgently.
- Fixforatleast30min.at4°C.
- Spinat2000rpmfor5min.Oncein70%ethanol,samplesmaybekeptforupto2weeks.
- DiscardsupernatantandresUSPendcellsin1mlPBS.Spin2000rpmfor5min.
- Repeatstep5twicemore.
- Add100µlribonuclease(100µg/ml,DNasefree,Sigma).Leaveatroomtemperaturefor5min.
- Add400µlpropidiumiodide(50µg/mlinPBS).Note:PIispotentiallymutagenicsoweargloves.BeforerunningthesamplesstainedforDNAcontentthecytometer"slinearity,resolutionanddoubletdiscriminationcapABIlityshouldbeverified.DNAQCkitisnecessaryandshouldbeorderedfromBectonDickinson(Cat.No.349523).Thekitiscomposedof:CEN-chickenerythrocytenuclei,usedinsettingcytometer"sphotomultipliertube(PMT)voltagesandamplifiergains,andprovidinginformationregardinginstrumentlinearityandresolution;CTN-calfthymocytenuclei,acyclingcellcomponentthatallowsassessmentofproperfunctionofdoubletdiscriminationmodule(DDM)orpulseprocessing.Pleasefollowtheinstructionsincludedinthekitforqualitycontrolandset-upofthemachine.
- AcquiredataonaFACScanorFACSCalibur,thecytometershouldbetriggeredonthePIsignal.Primarygateisonforwardscatter(FSC)againstrightanglelightscatter(SSC).
- Asecondarygateshouldbeplacedaroundthesinglecellpopulationonapulseareaversuspulsewidthdotplot.
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