Cell cycle analysis

۱٫ Fix cells by slowly adding ice-cold 70% ethanol and keep at at 4°C (or -20°C) for >4h. At this point
cells can be stored for months.
۲٫ Spin down the cells (500xg, 5 min) and wash the pellet once with PBS + 2%FCS. Cells fixed with
ethanol usually requires a longer centrifugation time. Be careful when removing the supernatant,
the pellet is easily disintegrated.
۳٫ Treat cells with RNase A. Add 100µl RNase A (for 1×10
6 cells) at a concentration of 1 mg/ml to the
cells and incubate at 37°C for 30 min.
۴٫ Stain the cells with 5-20µg PI in 0,5-1 ml depending on your cell amount. incubate for ≥۳۰ min at RT
protected from light.
۵٫ Analyze your sample on the Accuri, use the flow rate slow and make sure the flow stays under 500
events/s. Gate your cell population in the scatter plot and view the cell cycle as FL3-H using a linear
scale.
NOTE
PI also stains RNA, the RNase treatment is crucial for a nice cell cycle analysis.
PI can also be used as viability dye since PI does not cross the intact cell membrane but enters dead cells
that have damage plasma membranes and stains the RNA and DNA in these cells.
PI is a carcinogen and should be handled with care!

نوشتن نظر

نشانی ایمیل شما منتشر نخواهد شد. بخش‌های موردنیاز علامت‌گذاری شده‌اند *