Measuring Bone Marrow Hydrogen Peroxide

Bone marrow cells were harvested from both femurs of 6-week-old male C57Bl6 mice (n=3) as previously described^{26 (link)}. Basal hydrogen peroxide production was measured using the Amplex Red hydrogen peroxide /peroxidase assay (Invitrogen Molecular Probes, Eugene, OR) as per manufacturer’s instructions. Briefly, freshly isolated bone marrow cells were washed twice in Ringer’s solution, and plated (20,000 cells/well) in quadruplicate into wells of a 96-well plate containing 50 μl of Amplex Red reaction buffer (50 μM Amplex red, 0.1 U^.ml⁻¹ HRP) with or without 200 nM GEN or 1 nM E2, and incubated at 37°C continuously for 1 hour, at which an absorbance reading (560 nm) was taken. Data is expressed as amount produced (μM), which was corrected for non-specific hydrogen peroxide production by subtracting experimental values from values obtained from control wells not containing cells. Similar results were obtained in a repeat experiment performed in quadruplicate.