Detection of pDC extracellular traps (pETs) by immunofluorescence.

<p>Human pDCs (2 × 10⁵/well) were left unstimulated (Unstim) or stimulated for 4 hr with <i>A. fumigatus</i> hyphae (2 × 10⁵). pDCs associated with <i>A. fumigatus</i> hyphae showed evidence of ETosis. pETs were visualized by indirect immunofluorescence using primary antibodies against citrullinated histone H3 and the pDC marker CD123. Alexa Fluor 488- and 568-conjugated secondary antibodies were used for visualization of citrullinated histone H3 (green channel) and CD123 (red channel), respectively. DNA was stained with DAPI. Images were captured with a confocal microscope and a 60x oil immersion objective. Wavelengths of 405 nm (diode), 488 nm (Argon), and 543 nm (HeNe) were used to excite DAPI, Alexa Fluor 488 (and transmission images), and Alexa Fluor 568, respectively. Images were captured in separate passes to avoid cross talk and are presented as maximum intensity projections from Z-stacks. (A) Unstimulated pDCs. (B-D) Aspergillus-stimulated pDCs demonstrating pETs formation. The data are representative of three independent experiments.