U mwig3

Images were captured using a fluorescence microscope (BX51; Olympus) with a digital CCD camera system DP71 (Olympus). The filter sets U-MNIBA3 and U-MWIG3 (Olympus) were used for the GFP fluorescence and auto-fluorescence of the chloroplast, respectively. The fluorescence intensity of sfGFP was quantified by ImageJ¹.

Fluorescence images were obtained using an IX71 or IX83 Microscope (Olympus, Tokyo, Japan) equipped with a 100 W high-pressure mercury lamp and a cooled CCD camera, ORCA-R2 (Hamamatsu Photonics, Hamamatsu, Japan). A dichroic mirror block (U-MWIG3, excitation 530–550 nm, emission > 575 nm; Olympus) was used for the observation of CD31 antibodies and ethidium homodimers. For the observation of calcein-AM and CD41 antibodies, another dichroic mirror block (U-MNIBA3, excitation 470–495 nm, emission 510–550 nm; Olympus) was used.

Cells were grown at 30 °C in tryptone glycerol (TG) broth [1% tryptone, 0.5% NaCl, 0.5% (w/v) glycerol] supplemented with 50 μg/ml ampicillin. Overnight cultures were diluted 50-fold into fresh TG medium and incubated for 4 h at 37 °C. Cells were harvested by centrifugation at room temperature and resuspended in 100 μl of TMN buffer [50 mM Tris-HCl (pH-7.4), 5 mM glucose, 100 mM NaCl] and 100 μM Ser-FAM [l-serine 5(6)-carboxyfluorescein ester] (Eurofins Genomics Inc., Tokyo) was added. If necessary, non-labelled l-serine or taurine (final concentration: 1 mM) was added to TMN buffer. Cells were then incubated for 30 min at 37 °C, and washed once with TMN buffer. An aliquot of the cell suspension was spotted onto a MAS-coated glass slide (Matsunami Glass Inc., Osaka). Cells were then observed under a fluorescence microscope (Olympus IX71) equipped with a 100× oil-immersion objective lens. GFP and TagRFP were visualised using fluorescence mirror units, U-MNIBA3 and U-MWIG3 (Olympus), respectively. All images were recorded and processed by using a cooled charge-coupled-device camera ORCA-ERII (Hamamatsu Photonics) and the software Metamorph ver. 7.6 (Universal Imaging).