---++ Measuring intracellular ROS: ---+++ Protocol: 1.) Grow 5mL overnight cultures of the strains to be tested. 2.) The following day, dilute back the cultures 1:100 into fresh media. 3.) Grow the cells to mid-exponential phase (OD = ~0.5). At this point, you may want to add an ROS elicitor to your culture (ex, H2O2, K<sub>2</sub>TeO<sub>3, </sub> 4.) Stress ce Overnight culture was passaged 1 : 100 into fresh medium. Monitor the obsorbance to 0.2-0.4. Exposed to K<sub>2</sub>TeO<sub>3</sub> (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<sub>2</sub>HPO<sub>4</sub> , 2mM KH<sub>2</sub>PO<sub>4</sub>, 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
This topic: Lab
>
WebHome
>
ProtocolList
>
ProtocolsIntrocellularROSDetection
Topic revision: r2 - 2017-07-29 - DaciaLeon