Update Notice: A Simple Microplate Assay for Reactive Oxygen Species Generation and Rapid Cellular Protein Normalization

After official publication in Bio-protocol (https://bio-protocol.org/e3877), we noted some improvements in our protocol. The edits to be performed are the following:

1. The authors have improved background with a lower dye concentration in Step 7 in Figure 3 should read 35 µl rather than 50 µl.
   
   
   Figure 3. Protocol schematic. Cells are loaded with DCF-based probe, incubated for desired treatment period (e.g., 1 h or 24 h) before plate spectroscopy readout of DCF as an indication of ROS generation. To normalize DCF signal to cell population per well, a simultaneous acidic protein precipitation stage and cellular protein dye stage is performed before solubilization and spectroscopic readout of SRB. The TCA-SRB assay can be utilized to obtain relative cell populations for growth inhibition, cytotoxicity or to normalize other cell-based assay readouts.
   
   
2. In the Procedure Section, the volume of TCA-SRB in Step C2 is 35 µl. The Step C3 edits are the following: Empty plate into an appropriate corrosives waste container and wash with 200 µl 1% acetic acid. A second wash may be required if the background is higher than expected, particularly with automated pipetting devices that cannot completely remove liquid from the corner of the well.
3. In the Notes Section, Protocol is not efficient for suspension cells after further testing.
   “This protocol is intended for adherent cells; for more effective fixation of loosely adherent cells or suspension cells up to 50% w/v TCA may be utilized (Kim et al., 1996).” Is replaced with “This protocol is intended for adherent cell types.”