Figure 1.

Opsonized Candida upregulates cytosolic gal3 expression in neutrophils. Bone marrow neutrophils were stimulated with opsonized Candida SC5314 (A–C) or GFP⁺ OG1 (D,E) for 5 or 15 min. Zero minutes represent unstimulated cell controls incubated in serum-containing medium for 15 min. (A) Cells were cytospun, permeabilized, and stained for gal3 (red) and nucleus (blue) before confocal microscopic analysis for gal3 expression. DIC, differential interference contrast. (B,C) Neutrophils were stained with rat anti-gal3 and PE-goat anti-rat antibodies, fixed in 4% PFA, permeabilized, and then stained with Alexa 647-rat anti-gal3 antibody. The % of cells expressing cytosolic gal3⁺ (B) and surface gal3⁺ (C) in total cell population are shown in upper panels. The levels of cytosolic (B) and surface gal3 (C) expression are shown as mean fluorescence intensity (MFI) in lower panels. Data are presented as mean ± SD. *p < 0.05, as analyzed by Mann–Whitney test. n = 3. (D,E) Neutrophils were allowed to ingest GFP⁺ OG1 Candida (green) and stained for gal3 (red) and nucleus (blue) before subjecting to fluorescence microscopic analysis. (D) The intensity of fluorochromes along the white arrow in the merged image is shown in the histogram on the right. (E) The % of gal3⁺ area (red) that is not overlapped with Candida (green) in the whole gal3⁺ area (red) (180,760 µm² in average) at 5 and 15 min after encounter was analyzed by Metamorph software. Each dot represents one photo image. n = 5.