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Measurement of Green Fluorescent Protein Expression and DNA Content in Unfixed Cells

来自 : 蚂蚁淘

Reagents

Cellstobestudiedexpressinggreenfluorescentprotein(GFP).NotethatthesamecelltypewithoutGFPisneededascontrol.

Hoechst33342stocksolution(1mg/ml)(seerecipe)

12X75mmculturetubes

Vortexmixer

Waterbathat37^oC

Method

1.Countcells.

2.Placeapproximately10⁶cellsintoa12x15mmtesttubeandspinthemdownbycentrifugationfor5minat300xg.

3.Removesupernatantbyaspirationorrapiddecantingandadd500mlofthemediumthatwasusedforgrowingthecellstobestudiedpre-warmedto37^oCtothecellpellet.Mixgently.Add5mlofHoechst33342stocksolutionandmixagain.Incubateat37^oCfor45min.

TheoptimalHoechstdyeconcentrationandstainingtimefordifferentcelltypesvaryasdyeup-takedependsoncellularmetabolicrates;thus,bothhavetobedeterminedempirically.Ingeneral,dyeconcentrationsbetween1mg/mland10mg/mlandincubationtimesbetween20minand90minwillproduceDNAhistogramswithacceptablecoefficientsofvariation.BecauseHoechstDNAstainingisperformedonunfixedcells,itispossIBLetouseothernon-vitalDNAdyes,e.g.,PI,7-aminoactinomycinD(7-AAD),forconcurrentdeadcelldiscrimination.

PreparationofHoechst33342stocksolution:
Dissolve1mgofHoechst33342powder(Molecularprobes,Eugene,OR)in1mlofdistilledwater.Storeat2-8^oCprotectedfromlightforupto1month.

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发布于： 2018-12-26 阅读（176）

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