Sp5 2

Barley leaves with transformed epidermal cells were vacuum infiltrated with 0.01% Tween20 and subsequently mounted in the same buffer under a coverslip for confocal microscopy. Leica SP5-X, SP5-II and SP8 confocal laser scanning microscopes mounted with ×63 water immersion lenses with a numerical aperture of 1.2 were used. For detection and localization of the fluorophores, green fluorescent protein (GFP) was excited at 488 nm and detected between 500 and 540 nm, monomeric yellow fluorescent protein (mYFP) was excited at 514 nm and detected between 528 nm and 560 nm, while mCherry was excited at 543 nm (Leica SP5-II) or 587 nm using the super continuum white laser (Leica SP5-X) and detected between 600 and 640 nm. To limit signal bleed-through between channels, the measurements of each fluorophore were performed in independent tracks exciting only one fluorophore at a time.

Non-treated THP-1 cells and primary monocytes were washed and resuspended in PBS and laid on coverslips pretreated with poly-L-lysine (0.1 mg/mL) (Sigma) for 20 min at room temperature. PMA-treated THP-1 and monocyte-derived macrophages (MDMs) were grown on non-treated coverslips, washed in PBS, and then fixed with 4% paraformaldehyde in PBS. Intracellular PLSCR1 was detected with the 1E9 mAb in 1% BSA-PBS, supplemented with 0.1% Triton X-100 for permeabilization, and then stained using Alexa488-coupled secondary antibody. Cell surface PLSCR1 was detected either with the 1E9 mAb or with the rabbit pAb. Samples were examined under an epifluorescence microscope (Leica DMB) with a cooled charge-coupled device camera (Micromax 1300Y/HS; Roper Princeton Instruments), using a Plan APO 100X objective. Images acquisition was perfomed with MetaMorph 7.6 (Molecular Devices). After culturing BMDMs for at least 10 days in 10 ng/ml mCSF (Miltenyi Biotec), cells were plated on non-treated coverslips, subsequently fixed and permeabilized as described above, and then labeled with TRITC-phalloidin (Sigma) and rabbit anti-Iba1 (Wako Chemicals GmbH) and an Alexa 647 coupled-goat anti-rabbit-IgG (Molecular Probes). Images were acquired using a confocal microscope (Leica SP5II) equipped with a Plan APO 63x objective (N.A. 1.40, pinhole 1.0).