Download Orflo Application Protocol 12/2016 Propidium Iodide (PI)

Orflo Application Protocol
12/2016
PropidiumIodide(PI)-CellCycleStainingProtocol
Reagents:
• PropidiumIodidesolution(1mg/ml,e.g.LifeTech.P3566)
• 70%Ethanol(e.g.SigmaE7023,inPBSordistilledwater)-Storeat-20°C
• IcecoldCa2+-freeandMg2+-freePBSwithMoxiFlowReagent
o PhosphateBufferedSaline(PBS,e.g.LifeTech#10010-23)
o 20µl/mLMoxiCyteFlowReagent(OrfloTechnologies,MXA080)
• DNAse-freeRNAseA(e.g.,SigmaR6513)dissolvedto1mg/ml CellCycleSynchronization(SerumStarvation):
1. Removeoldmediaandreplacewithserumfreemedia(NOTE:Somecell
linesrespondpoorlytoserum-freemedia.Theselinesmightrequirea
gradualintroductiontoserum-free(24hrsin1%FBS,24hrsin0FBS)
2. Toreleasecells,passageasappropriateandadd10%FBS.Forcellcycle
arrestdrugaddition,adddrugtomediaPRIORtoaddingcellstoensure
propermixing/dilution
EthanolFixationProtocol
1. Pelletcells(300xg,5min)andremovesupernatant.Atthisstageitiscritical
toachieveasingle-cellsuspension.Notes:
i. Ifyouaredetachingadherentcells:
1. Leavethecellsinthedetachmentmedia(e.g.Accutase)for
15+minutes.
2. Attheendoftheincubationperiod.Pipettetrituratethecells
inthatdetachmentmediatobreakupclusters.
3. Vortexthedetachmentmedia/cellsuspensiongently(e.g.3
setting)for~30secondstofurtherbreakapartthecluster
ii. Forsuspensioncells:
1. Resuspendthecellsinadetachmentmedia(e.g.Accutase)
pipettingtodispersethecells
2. Leavethecellsinthedetachmentmedia(e.g.Accutase)for
15+minutes.
3. Attheendoftheincubationperiod.Pipettetrituratethecells
inthatdetachmentmediatobreakupclusters.
4. Vortexthedetachmentmedia/cellsuspensiongently(e.g.3
setting)for~30secondstofurtherbreakaparttheclusters.
2. WashthecellstwiceinPBS(300xg,5min,4°C).Countthecellswhen
finished.
a. Note1:Recommendatotalof5e5cellssothatthere-suspensionis
1e6/ml(whenadding0.5mlfinalre-suspensionlater).
b. Note2:PutPBSoniceduringcentrifugationstokeepcold.
c. Note3:Pipettetriturate10-20xwhenre-suspendingpellettoensure
singlecellsuspension.Preferredapproachisadd~1mLPBStopellet
ORFLO Technologies
Ketchum, ID
[email protected]
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www.orflo.com
Orflo Application Protocol
12/2016
PropidiumIodide(PI)-CellCycleStainingProtocol
3.
4.
5.
6.
7.
andusea
1mLpipettetotriturate.Thenaddremaining4mlPBSforwash.
Invert3x.
Re-suspendthepelletinapproximately500ulofice-coldPBS.Pipetwith
1000µLpipette,upanddown,20times.Itisimportantthatthisbeagood
single-cellsuspensionatthispoint,orthecellswillbefixedasclumps.
Aliquot4.5mLoficecold70%ethanoltoa15mLcentrifugetube.
Holdethanoltubeandcelltubeinacoldpackandvortexgently.
Add.5mlcellsdrop-wisetothe70%ethanoltube(whilevortexing)usinga
100uLpipette(maxsizetoensuredropvolumeissmall).
PlaceinFreezerfor2hrto4days
PropidiumIodideStainingProtocol
1. Centrifugeat800xg,10°C,5minwithBRAKEOFF.Removetheethanol.
2. Re-suspendin1mlice-coldPBSwith20xpipettetrituration(1000uLpipette)
3. Add4mLice-coldPBSandinvert3x.
4. Centrifugeat700xg,10°C,5minwithBRAKEOFF.Removethesupernatant.
5. Re-suspendin1mlice-coldPBSwith20xpipettetrituration(1000uLpipette)
6. Add4mLice-coldPBSandinvert3x
7. Centrifugeat600xg,10°C,5minwithBRAKEOFF.Removethesupernatant.
8. Re-suspendthepelletedcellsin1mlofPBS.UseMoxiGOtodetermineacell
concentrationandadjustthecellconcentrationto2e5cells/ml.
9. Add20ul/mlPropidiumIodide(1mg/ml)to1mlofthe2e5/mlcell
suspension(finalPIconcentrationof20µg/ml).
10. Add20uLof10mg/mlRNAseandincubateat37°Cfor30minutes.
11. AnalyzeonMoxiGOusingtheCellCycleAssay.
ORFLO Technologies
Ketchum, ID
[email protected]
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855-TRY-MOXI
www.orflo.com