Cc12 digital color camera

For immunofluorescence, cells were cultivated as described above in LB or AB minimal medium to early stationary phase, harvested and adjusted to an OD₆₀₀ of 2 in PBS buffer. Cells were fixed in 2.7% paraformaldehyde and 0.01% glutaraldehyde for 30 min at 4°C. Cells were washed in PBS and GTE buffer containing 50 mM glucose, 10 mM EDTA, and 20 mM Tris. Permeabilization of the membrane was achieved by incubation in PBS buffer containing 5 mM EDTA and 5 mg/ml lysozyme at 4°C for 5 min followed by PBS washing and incubation in PBS containing 0.1% Triton X-100 for 5 min at room temperature. Triton X-100 was removed by PBS washing and cells were blocked with PBST [PBS containing 0.1% tween 20 (vol/vol)] containing 3% BSA (wt/vol) for 1 h at room temperature. Cells were again washed in PBST and incubated with primary antibody solution in PBS, 0.1% BSA for 1 h at 30°C. After washing with PBST, cells were incubated with secondary antibody solution in PBST for 1 h at 30°C. Finally, cells were washed three times in PBST, resuspended in 200 μl PBS and mounted onto agar pads containing 0.5% low-melting agarose. Cells were imaged with an Olympus BX51 microscope using a U-UCD8 condenser and an UPlanSApo 100XO objective and images were taken with a CC12 digital color camera (all components by Olympus, Hamburg, Germany).

Paraformaldehyde-fixed cells from the phagocytosis assay were centrifuged at 200 g for 5 min and resuspended in PBS containing 0.1% Tween 20 and 2.5 µg. mL⁻¹ TRITC-conjugated phalloidin (Sigma-Aldrich). Cells were incubated with phalloidin for 40 min at room temperature, protected from light. Excess phalloidin was removed by centrifugation and cells were resuspended by pipetting up and down in buffered glycerol containing 0.5 µg/mL DAPI (Sigma-Aldrich). Cells were then visualized on a slide in a BX51 fluorescence microscope with a U‐UCD8 condenser, a U‐LH100HGAPO burner, a U‐RFL‐T power supply, a 63X/1.4 NA oil objective and a 450–490 nm excitation/500–550 emission bandpass filter (Olympus, Tokyo, Japan). Images were collected with a CC12 digital color camera and the Cell Imaging Software (Olympus) and composite figures were prepared using ImageJ (Schneider et al., 2012 (link)) and Photoshop CS5 (Adobe Systems, San Jose, CA) software.