Cxp acquisition

Analyses were performed on three Cytomics FC500 flow cytometers (Beckman-Coulter, Villepinte, France). To limit background noise from dust and crystals, all three instruments were operated using 0.22-μm filtered sheath fluid (Isoflow™; Beckman-Coulter). CXP ACQUISITION and CXP ANALYSIS software packages (Beckman-Coulter) were used for data acquisition and analysis, respectively. Arabidopsis protoplasts of leave 5 were immersed in 5 μM FDA (Sigma; in MES buffer, pH 6.1) for 20 min at room temperature in the dark, and then washed three times with MES buffer (pH 6.1). Cells were stained with Annexin V using the Annexin V-FITC fluorescence detection kit (BD Biosciences, San Jose, CA, USA), in accordance with the manufacturer's instructions. Briefly, cells cultured on cover slips, and then washed twice with PBS. The slides were examined and photographed with a Nikon Eclipse TE 2000 U motorized inverted microscope (Nikon Corp., Tokyo, Japan). The apoptotic index was calculated as the percentage of cells stained positive for Annexin V. A total of 100 cells were counted in each experimental group in three independent experiments and results arethe mean proportion of apoptotic cells in sixscanning electron micrographs.

Analyses of labeled samples were performed on a Cytomics FC500 flow-cytometer (Beckman Coulter) as previously described (Robert et al., 2009 (link)). Briefly, after standardization of the protocol with a blend of monodisperse fluorescent beads of three diameters (0.5, 0.9 and 3 μm, Megamix, Stago, Biocytex, Marseille, France), optimal instrument settings and the MP region were defined. Megamix beads were run before starting each analysis in order to control and, eventually, to adjust FCM-settings. Forward (FS) and side (SS) scatter parameters were plotted on logarithmic scales to best cover a wide size range. PMPs were gated in the MP window and defined as single CD61 (or CD41)+ events or dual-positive phosphatidylserine (PS)⁺/CD61 (or CD41)⁺ events, as seen in dual-color fluorescence plots after staining with annexin V-FITC and CD61 (or CD41)-PE. Single staining controls were used to check fluorescence compensation settings and to set up positive regions. Each tube was run for 1 min at medium flow-rate, with a maximum delay of 30 min after the end of staining.
To limit background noise from dust and crystals, flow cytometric analyses were performed using a 0.22 μm-filtered sheath fluid (Isoflow^TM, Beckman Coulter). CXP ACQUISITION and CXP ANALYSIS software packages (Beckman Coulter) were used for data acquisition and analysis, respectively.