Life Technologies
CellROX Green
C10444
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ROS production after cell cycle arrest. Ready-to-use kit, there is no optimalisation needed for single colour staining (we did not even need further optimalisation for multicolor FC analysis). Fixable (we used PFA). Before permeabilization, think about optimalising the detergent/permeabilizing agent concentration (we used Triton X-100 and saw a loss of signal).
Flow cytometry
Cell culture - HCT116 wt
Add proper amount of reagent directly to the cell culture dish, incubate 30 min/ 37 C (5 % CO2). Same for treated and untreated cells. ROS generation might be stopped by fixing with PFA (needed especially when using positive control). Always use controls! Positive ctrl - menadion or its derivates (100 uM/ 10 min/ 37 C). Negative ctrl - acetylcysteine (or another scavenger).
There is no need to add the reagent directly to the dish, cells might be detached, washed and resuspended in PBS (10^6 per 100 ul PBS). This will significantly reduce the amount of reagent without influencing the results.
Nice separation between autoflorescence and positive ctrl.
Ease of use
N/A
If there is some simple method for ROS detection needed, I would recommend CellROX Green both for flow cytometry and fluorescence microscopy.
Product Name: CellROX® Green Reagent, for oxidative stress detectionSupplier Page