Fig. 1

Nonlytic exocytosis of C. neoformans by neutrophils demonstrated by time-series images and a 3D reconstruction of z-stack images. a Time-series images. C. neoformans H99 cells labeled with FITC were incubated with neutrophils, which were prestained with anti-Gr-1 (pseudocolored red) and anti-F/80 (pseudocolored blue), in a glass-bottomed dish with a precoated monolayer of mouse brain endothelial bEnd.3 cells in the presence of anti-capsule antibody E1. After 1 h of incubation, nonadherent cells were removed. Multichannel time-lapse fluorescence images were taken to record the dynamic process of nonlytic exocytosis of C. neoformans by the neutrophil (marked by an arrow), capturing one frame every 30 s for 3 h or until the occurrence of expulsion. At the start of image acquisition, the neutrophil contained two fungal cells. Forty-six minutes later, the phagosome fused with the cell membrane. The two fungal cells (marked by arrowheads) escaped from the neutrophil within 1 min. b A 3D image reconstruction. To verify the expulsion of C. neoformans, z-stack images were taken at the end of the image acquisition period (81 min after the microscopy observation began) for the reconstruction of a 3D image of neutrophils and fungi. The neutrophils did not contain fungi; the two expelled fungal cells were located beside the “empty” neutrophil. Bar, 5 μm. c Flow cytometry analysis of neutrophils. Neutrophils purified from bone marrow were stained with Alexa Flour 647 anti-F4/80 and Alexa Flour 555 anti-Gr-1 or isotype control antibodies. Based on the flow cytometry analysis, approximately 90% of cells were F4/80⁻Gr-1⁺ (neutrophils), and less than 1% of cells were F4/80⁺Gr-1⁻ (resident monocytes) or F4/80⁺Gr-1⁺ (inflammatory monocytes), indicating that the vast majority of Gr-1-positive cells in the cell preparation were neutrophils. Representative results from 3 repeated experiments