NADPH oxidase activity in stimulated human and murine phagocytes in the absence and presence of BK channel inhibitors. (A) Respiratory burst by rhodamine generation in PMNs and monocytes assayed in either human or mouse whole blood by flow cytometry. Whole blood (100 μL) was loaded with DHR after red blood cell lysis. Generation of rhodamine was measured in FL-1 channel (rhodamine fluorescence intensity). To ensure that the gated cells were specifically either PMNs or monocytes, staining with the indicated antibodies and controls was performed. One of 2 independent experiments is shown. (B) Respiratory burst in BMDMs. BMDMs were differentiated from WT bone marrow. Cells stained positive for the macrophage marker F4/80, but not for the isotype control antibody. PMA stimulation did not evoke generation of reactive oxygen species, as measured either by rhodamine generation (second panel from right) or ferricytochrome c reduction (right panel). A representative of 3 independent experiments is shown. (C) Effect of IbTX and paxilline on rhodamine production of human PMNs and monocytes assayed in whole blood by flow cytometry. A total of 100 μL of whole blood was loaded with DHR after red blood cell lysis. Samples were preincubated with IbTX and paxilline, respectively. After 10 minutes, cells were stimulated with either PMA or opsonized zymosan. Generation of rhodamine was measured in FL-1 channel and is reported as rhodamine mean fluorescence intensity (MFI). Neither IbTX nor paxilline inhibited rhodamine production in human PMNs and monocytes (n = 2).