Hcx pl fluotar 100 1.30 oil objective

Glycotyping with a pair of GFP-tagged phage proteins including A006_gp17 (to identify rhamnose) and CBDP35 (to identify N-acetylglucosamine) was performed as previously described [32 (link)]. Briefly, Listeria cells from log phase cultures (OD600nm = ~0.5) were harvested by centrifugation (10,000 g, 1 min), and resuspended in 1/5 volume of PBS (pH 7.4). The cell suspension (100 µL) was incubated with 5 µL of 1 mg/mL of the GFP-fused proteins and incubated for 5 min at room temperature. The cells were centrifuged, washed twice in PBS and finally resuspended in PBS. The samples were transferred onto a glass slide with a cover slip and examined by a confocal laser scanning microscope (Leica Microsystems GmbH, Germany) equipped with a HCX PL FLUOTAR 100 × 1.30 oil objective. Image analysis was performed in Leica Suite Software (Bitplane AG, Zurich, Switzerland). For ease of visualization, contrast in red and green channels was enhanced. Each strain was tested in at least two independent trials.

For microscopy, 5 μl of Listeria suspensions labeled with fluorescent proteins as described above or 5 μl of Listeria coupled to paramagnetic beads were transferred onto a glass slide with a cover slip. Imaging was performed on a TCS SPE confocal system (Leica Microsystems GmbH, Germany) equipped with an HCX PL Fluotar 100 × 1.30 oil objective. GFP and CFSE were excited at λ of 488 nm, and red fluorescent protein (RFP) was excited at λ of 532 nm. For 3D reconstructions, z-stacks of 20 μm with a z-slice thickness of 0.5 μm were imaged. Image analysis was performed in Imaris Software (Bitplane AG, Zurich, Switzerland), and for ease of visualization, the contrast in red and green channels was enhanced.