Measuring intracellular ROS:
Protocol:
Overnight culture was passaged 1 : 100 into fresh medium.
Monitor the obsorbance to 0.2-0.4.
Exposed to K
2TeO
3 (0.5 µg/ml) for 30 min.
Centrifuge at 3,000 g for 5 min at 4°C , cells were washed with ice chilled saline phosphate buffer (137 mM NaCl 2.7 mM KCl, 10 mM Na
2HPO
4 , 2mM KH
2PO
4, pH 7.3) and resuspended with two volume of the same buffer, conducted at 4°C .
Cells were incubated with 100 uM (50 µg/ml) 2’,7’-dihydrodichlorofluorescein diacetate at 37°C for 60 min in water bath.
Centrifuge at 3,000 g for 5 min at 4°C. Wash once with ice chilled PBS. Centrifuge and put on ice before run flow.
Fluorescence intensity was determined by flow cytometry.
-- Main.XueZhang - 25 May 2017
Topic revision: r1 - 2017-05-25 - 20:10:52 - Main.XueZhang