Cxp analysis

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.

PBMCs concentrated to 5 × 10⁶ cells/mL were stained with fluorescent antibodies directed against the surface markers CD14, CD16 and HLA-DR (CD14:PerCP, CD16:FITC, HLA-DR:PE) or IgG controls (BD Biosciences, Thermo Fisher Scientific, Sunnyvale, CA, USA). Fluorescent cells were measured by flow cytometry (Beckman Coulter, Cytomics FC 500 MPL, Miami, FL, USA). After compensation, the mean fluorescence intensity (MFI), frequency of cells positively stained and resultant populations based on the combination of markers were analysed using CXP Analysis (Beckman Coulter, Brea, CA, USA).

YD10B and HSC2 cells (5 × 10⁵) were grown in 100-mm dishes incubated at 37 °C overnight. The cells were then treated with the O. octandra extract for the indicated period. Afterwards, the cells were stained using an annexin V-fluorescein isothiocyanate (FITC) kit (#556547, BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. Briefly, both types of cells were washed by cold PBS and then the cells were incubated with FITC annexin V and/or PI in 1X Binding Buffer for at room temperature 15 min. The stained cells were then analysed by flow cytometry. For bar graphs, the fraction of early apoptotic cells was measured by detecting cells stained only with annexin V. A FC500 series CXP cytometer and CXP analysis (Beckman Coulter, USA) were used for measurement and data analysis, respectively.