Figure 3.

Detection of phagosomal O₂^·− in Nox2-KO microglia engulfing zymosan. A, Cultured Nox2-KO microglia were incubated for 30 min with zymosan (Zym) in the absence or the presence of DPI, before addition of NBT to the culture for 15 min. TL, Transmitted light. DIC, Differential interference contrast. White and black arrowheads indicate extracellular and engulfed yeast particles, respectively. The dark blue formazan deposit overlying the phagosomal membrane is not observed in DPI-treated cells. B, Red fluorescence emitted by oxidized hydroethidine (Ox-HE) in a Nox2-KO microglial cell (same cell as in Fig. 2B) after a 45 min incubation with zymosan in the presence of cell-permeant hydroethidine. The DNA of the cell and engulfed zymosan were counterstained with Hoechst 33342 (blue fluorescence converted to green), and the cell nucleus and the plasma membrane are outlined in yellow and white, respectively. Ox-HE fluorescence is colocalized with the DNA of engulfed zymosan (confocal microscopy). Scale bars, 10 μm.