Spinsr10 ixplore

Immunofluorescence (IF) experiments were performed as previously described (Jiu et al., 2019 (link)). Briefly, cells were fixed with 4% PFA in PBS for 15 min at room temperature (RT), washed three times with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. Cells were then blocked in PBS supplemented with 5% BSA. Both primary and secondary antibodies were applied onto cells and incubated at RT for 2 h. Alexa-conjugated phalloidin was added together with secondary antibody solutions onto cells. Alexa 647-wheat germ agglutinin (WGA) (#W32466, Thermo Fisher Scientific) was used to visualize the plasma membrane. All IF data were obtained with Olympus SpinSR10 Ixplore spinning disk confocal microscope with UplanApo 100×/1.5 Oil objective (Olympus Corporation, Tokyo, Japan). The pixel size was optimized properly to achieve the maximum resolution which was calculated to be 65 nm. For detection and measure of the cytoplasmic CAV-1 tagged vesicles, the “Spots” tool of Imaris 9.2 (Bitplane, Zurich, Switzerland) was used with the configuration defined as 2 μm for estimated XY diameter. The numbers and sizes of spots were calculated subsequently. For detection and measure of the lamellipodia width, a plot profile perpendicular to the plasma membrane was made by using imageJ, and the peak zone was defined as the width.

Briefly, 5 × 10⁵ live yeasts were added to 5 × 10⁵ macrophages or neutrophils in μ-Slide 4-well chambers (ibidi GmbH, Germany) immediately prior to imaging. Live video microscopy was conducted using an Olympus SpinSR10 Ixplore under the same conditions described for the phagocytosis assay. Images were taken every minute over a 6-h period at a ×63 or ×100 magnification and analyzed using imageJ analysis software. Results are expressed as engaged cells (the percentage of cells engulfing or adherent to fungal cell) and phagocytic index (the total number of fungal cells taken up per 100 cells). Data were obtained in triplicate from at least 3 separate experiments by analyzing at least 200 macrophages per well.