Axioskop 3 channel confocal microscope

Phagocytosis of fluorescently labelled beads was determined by adding a 10-fold excess of fluorescent beads (0.7–0.9 µm diameter size) to a cell climbing piece. To measure the internalization of beads, they were incubated with macrophages for 3 h at 37 °C, and cells were then washed 3 times within ice-cold PBS. After fixing in 4% paraformaldehyde for 30 min and permeabilizing using 0.5% Triton X-100 for 10 min, the cells were incubated in Alexa Fluor 488-phalloidin (Green) for 30 min at room temperature. DAPI counterstaining was performed for 20 min to visualize the DNA. Fluorescence was detected using an Axioskop 3-channel confocal microscope (Zeiss710), and the fluorescence intensity was quantified using Image J software. Amounts of fluorescent beads/cell were calculated using IP-lab software. At least three separate fields from triplicate wells were analysed per experiment for each genotype.

Macrophages were cultured on coverslips for 24 h, incubated in media lacking FBS for 3 h or 6 h, and then fixed with 4% paraformaldehyde in the presence of 1 U/mL rhodamine-phalloidin for 30 min at room temperature. Then, coverslips were mounted on slides and observed using an Axioskop 3-channel confocal microscope (Zeiss710). Images were obtained via z-scanning and were analysed using Zen2010 software. At least three separate fields from triplicate wells were analysed per experiment for each genotype.