Eclipse te2000 e inverted fluorescence microscope

To generate the BiFC constructs, the full-length cDNA sequences of MxERF4 and MxFIT were cloned into the pSPYNE-35S and pSPYCE-35S vectors^{72 (link)}. The primers used for the BiFC assays are listed in Supplemental Table S1. Coexpression was observed in tobacco (Nicotiana tabacum) leaves as described by Schütze et al.^{45 (link)}. The fluorescence of the fusion proteins was detected 3 days after Agrobacterium infiltration. Fluorescence images were acquired using a Nikon D-ECLIPSE C1 spectral confocal laser-scanning system. YFP and brightfield images were generated by excitation at 488 and 543 nm, respectively. A Nikon ECLIPSE TE2000-E Inverted fluorescence microscope was used for the fluorescence analysis.

Disaggregated cells were seeded at a density of 1 × 10⁵ cells/ml in 8 mL of complete RPMI 1640 medium and the formation of aggregates was examined at 72 hours of culture by phase contrast with a 10 × objective. To examine the effect of divalent cations on aggregate formation, cells were cultured in the presence or absence of 1 mM EDTA (Sigma-Aldrich) or EGTA (Sigma-Aldrich) for 24 h. For antibody-blocking experiments, cells were placed in 96-well culture plates at a density of 0.5–1 × 10⁶ cells/ml in 100–150 μl/well of complete RPMI 1640 medium containing 10 µg/ml anti-CD18 (Biolegend), anti-CD62L (BioLegend) mAbs or mouse IgG isotype control (BioLegend) and then incubated at 37°C for 2 h. Photomicrographs were taken using an ORCA-ER monochrome cooled-CCD camera (Hamamatsu, Hamamatsu, Japan) coupled to an Eclipse TE2000-E inverted fluorescence microscope (Nikon, Tokyo, Japan) and NIS-Elements AR imaging software and the number of aggregates containing more than 20 cells per field was counted.

Binary vectors were transformed into the Agrobacterium tumefaciens strain EHA105, and agrobacterium infiltrations of N. benthamiana leaves were performed essentially as described by Johansen and Carrington (2001). The agrobacterium mixtures also usually included bacteria containing the pGD-P19 plasmid to minimize host gene silencing [72] (link). The lower epidermal cells of agro-infiltrated leaves were assayed by CLSM at 3 dpi. Fluorescence analysis was performed using a Nikon ECLIPSE TE2000-E inverted fluorescence microscope equipped with a Nikon D-ECLIPSE C1 spectral confocal laser scanning system. RFP was excited using a 543 nm laser and imaged using the META detector set for 570–600 nm. DAPI fluorescence was excited with a filter set consisting of an emission filter of 435–485 nm and a 408-nm laser. The images were captured and processed with ECLIPSE EZ-C1 3.00 FreeViewer software, and data were captured as single optical sections.