Plan apochromat 63 1.40 oil dic m27 objective lens

J774/mVenus and J774/mVenus-MORN2 cells were plated onto the center of 35-mm-diameter glass-bottom dishes at a density of 0.75×10⁶ cells and labeled overnight at 37°C with 50 µg/ml CF640R-dextran (MW 10,000 Da; Biotium, Fremont, CA, USA), after which the labeling medium was removed. Subsequently, the cells were chased for 5 h, pre-incubated with or without 10 µM DPI for 30 min, washed with ice-cold Hank's balanced salt solution (HBSS) and incubated for 30 min on ice in the presence of an approximately 30-fold excess of Texas Red-conjugated zymosan. Next, the cells were maintained at 30°C for 5 min to initiate phagocytosis, washed with ice-cold HBSS, incubated with anti-zymosan antibodies for 20 min on ice and maintained with Alexa 405-conjugated anti-rabbit secondary antibodies to stain extracellular zymosan. Finally, the cells were incubated in HBSS-containing cytochalasin B (20 μM final concentration) for 15 min at 30°C. Images were captured on an LSM780 laser-scanning microscope with a Plan-Apochromat 63×/1.40 oil DIC M27 objec­tive lens (Carl-Zeiss, Oberkochen, Germany) under low temperature conditions (6°C). More than 40 phagosomes were counted for each experiment and categorized as CF640R-dextran-positive or unlabeled phagosomes based on the presence or absence of CF640R fluorescence signal.

FRET experiments were accomplished using a laser scanning confocal microscope (Zeiss LSM 710) with a Plan-Apochromat 63 × /1.40 Oil DIC M27 objective lens (Zeiss). A 543 nm laser was used for acceptor (mKate-FAK) photobleaching within a region of interest (ROI). One frame was acquired as a pre-bleaching control, and the ROI was bleached within one frame. Zeiss Zen 2010 software was used for FRET analysis.