AA and SS RBC contain several NOX isoforms. (A) NOX isoforms are detectable in AA and SS RBC. Western blotting of total RBC lysates reveals the presence of the NOX isoforms NOX1, NOX2 (gp91), NOX4, and NOX5 in both AA and SS RBC. NOX3 could not be detected. Control samples for the different NOX isoforms were human WBC lysate (NOX1 and NOX2), human kidney lysate (NOX4), and human testis lysate (NOX5). (B) NOX1 and NOX2, but not CD45, are detected in AA and SS RBC, indicating that the presence of NOX isoforms in the RBC lysate is not due to WBC contamination. (C) NOX1 and NOX2 are detected by immunofluorescence in intact WBC, as expected, as well as in intact RBC of whole SS blood samples, by multiparameter high-speed cell imaging in flow. (D) Up to 80% of sickle RBC have positive stain for NOX1, with 6% of the erythrocytes having a mean pixel value (expressing the MFI) of NOX1 >50 (arbitrary units), at the level of positivity for NOX1 in WBC within the same sample. NOX2 positivity appear less intense for the sickle erythrocytes in comparison with WBC, with only 2% of the RBC staining as strongly positive as the WBC.