How do you detach cells for flow cytometric analysis when using Cell Meter™ Fluorimetric Intracellular ROS Activity Assay?

I want to use your assay, Cat# 22903, with FACS. When should I add the dye to my sample? When should I detach my cells from the cell plates? And, what technique should I use to detach my cells?

When using Cat# 22903 we first treat our cells with TBHP for 30 minutes to induce ROS. Then we stain the sample with ROS Brite™ 670 at 37 °C for 60 minutes. Next, we use trypsin to dissociate adherent HeLa cells from our cell culture plates, and then we monitor the fluorescence fluorimetrically at Ex/Em = 650/675 nm. Flow cytometeric analysis of HeLa cell suspensions show a 10-20 fold increase in TBHP treated samples compared to the corresponding control.

In the above technique we applied the ROS assay to adherent cells first before detaching the cells. However, if you choose to detach your cells first and then apply the ROS assay, your results will show a similar response.