NEM-based alkylation for measuring H2O2 levels in cell cultures.

The exact cell number of Chlamydomonas cells grown to 1–3×10⁶ cells·mL⁻¹ was determined and the volume to harvest 2.5×10⁷ cells was calculated. For rapid “trapping” of probe oxidation, 50-mL Falcon tubes were preloaded with one fourth of the final volume with 100-mM MES–Tris (pH 7.0) buffer containing 40-mM NEM. Chlamydomonas cells were harvested in prefilled Falcon tubes by centrifugation at 3,150g for 3 min at room temperature and subsequently resuspended in 500 µL of 100-mM MES–Tris (pH 7.0) buffer containing 40-mM NEM to a final cell density of 5×10⁷ cells·mL⁻¹. 200 µL (corresponding to 10⁷ cells) of cell suspension was transferred to a 96-well plate. For calculating OxD, fully oxidized (with diamide) and reduced (with DTT) samples were added and probe oxidation was measured following the protocol described above with 15 measurements per biological sample to reduce technical variability. To verify efficient trapping of the sensor oxidation state with NEM, roGFP2 fluorescence recordings using 0.1-mM H₂O₂ were performed as described above. Twenty microliters of NEM (200-mM stock) was added to a final concentration of 16.67-mM NEM per well at the indicated time differences.