Solutions

• 70%ethanol
• ribonuclease(100µg/mlDNasefree,Sigma)
• propidiumiodide(50µg/mlinPBS)

Procedure

1. Harvestcells.Spinat1200rpmfor5minutes.
2. Discardsupernatant;add1ml"fridgecold"70%ethanolto1x106cellswhilevortexingthecellpelletgently.
3. Fixforatleast30min.at4°C.
4. Spinat2000rpmfor5min.Oncein70%ethanol,samplesmaybekeptforupto2weeks.
5. DiscardsupernatantandresUSPendcellsin1mlPBS.Spin2000rpmfor5min.
6. Repeatstep5twicemore.
7. Add100µlribonuclease(100µg/ml,DNasefree,Sigma).Leaveatroomtemperaturefor5min.
8. 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.
9. AcquiredataonaFACScanorFACSCalibur,thecytometershouldbetriggeredonthePIsignal.Primarygateisonforwardscatter(FSC)againstrightanglelightscatter(SSC).
10. Asecondarygateshouldbeplacedaroundthesinglecellpopulationonapulseareaversuspulsewidthdotplot.

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