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7 protocols using cxp analysis

1

Quantifying Arabidopsis Leaf Protoplast Apoptosis

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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.
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2

Multivariate Survival Analysis Protocol

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Kaplan-Meier survival curves were generated with GraphPad Prism 5 (GraphPad Software Inc, La Jolla, CA). Flow cytometry data were analyzed with CXP Analysis (Beckman Coulter Inc.), Diva (BD Biosciences) and FlowJo software (Tree Star Inc, Ashland, OR). Statistical analysis including the student t-test of the flow cytometry data, the log-rank analysis of the survival curve was performed with Excel (Microsoft Inc, Seattle, WA), GraphPad Prism 5, and SAS 9.1 (SAS Institute Inc. Cary, NC). Statistical data with replicates were represented by the average values and the standard deviation.
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3

Comprehensive Immune Cell Profiling

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Fc receptors were blocked with mouse Fc block and the dead cells were detected using Fixable Viability Dye eFlour™ 506 or 510 (eBioscience) before cell surface staining. For Breg staining, CD19-FITC or PE/Cy5, CD24-PE, CD38-PE/Cy7, CD1d-PE, and CD5-FITC mAbs were used. For intracellular IL-10 staining, cells were stained with CD19-PE/Cy5 or APC mAbs. Cells were washed, fixed with IC Fixation Buffer (eBioscience), permeabilized with Permeabilization Buffer (eBioscience), and stained with IL-10-PE. For Treg staining, cells were stained with combinations of CD4-FITC and CD25-PE/Cy5.5 or APC mAbs, fixed and permeabilized with Fixation/Permeabilization solution (eBioscience) and Permeabilization Buffer, and stained for detection of intracellular Foxp3-PE mAbs. For apoptotic cell detection, cells were washed twice with cold PBS and then resuspended in 1× Binding Buffer (BD Biosciences), and then the cells were stained with CD4-FITC, APC Annexin-V, and 7-AAD and incubated for 15 min at RT in the dark. Last, 400 μl of 1× Binding Buffer was added. Data were acquired using Cytomic FC500 or Cytoflex (Beckman Coulter) and analyzed using CXP Analysis and Cytexpert (Beckman Coulter).
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4

Phenotypic Analysis of Human PBMC Subsets

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Fresh PBMCs were stained with the following conjugated anti-human monoclonal antibodies (mAb): fluorescein isothiocyanate (FITC)–conjugated anti-CD21, energy-coupled dye–conjugated anti-CD19, allophyocyanin (APC)–conjugated anti-IgM, and phycoerythrin (PE)–Cy7–conjugated anti-CD27 (Beckman Coulter). PBMCs were also stained with PE-conjugated anti–FCRL-1, anti–FCRL-2, anti–FCRL-3, anti–FCRL-4, and anti–FCRL-5 mAb. We prepared these mAb as described previously (31 (link)–34 (link)). All of these FCRL-specific mAb were authorized as reference antibodies for FCRL proteins 1–5 (CD307a–e) in previous international human leukocyte differentiation antigens workshops (33 (link),35 (link)). We also used PE-conjugated anti–FCRL-4 mAb (clone 413D12) from BioLegend. PE-conjugated IgG1 (Beckman Coulter) was used as an isotype control. Flow cytometry was performed on a Navios flow cytometer (Beckman Coulter) and analyzed using CXP analysis (Beckman Coulter) and FlowJo software (Tree Star).
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5

Flow Cytometric Analysis of Platelet Microparticles

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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 (IsoflowTM, Beckman Coulter). CXP ACQUISITION and CXP ANALYSIS software packages (Beckman Coulter) were used for data acquisition and analysis, respectively.
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6

Phenotyping PBMCs by Flow Cytometry

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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).
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7

Apoptosis Analysis of O. octandra Extract

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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.
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