Computational protocol: Cross sectional study of soluble selectins, fractions of circulating microparticles and their relationship to lung and skin involvement in systemic sclerosis

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Protocol publication

[…] MPs, here defined as particles smaller than 1 μm, were measured directly in platelet poor plasma []. In brief, a panel of cell-specific monoclonal murine antibodies was applied to label MPs originating from platelets (CD42a), leukocytes (CD45) and endothelial cells (CD146). The antibodies were used in the following formats and final concentrations after titration experiments: anti-CD42a-FITC (IgG1, 32 ng/mL, Becton-Dickinson, clone ALMA16), CD45-PE (IgG1, 65 ng/mL, Becton-Dickinson, clone HI30) and anti-CD146-FITC (IgG1, 262 ng/mL, AbD Serotec, clone OJ79c). Labeling of MPs by specific antibodies was compared to MPs labeled by isotype-matched control antibodies (IgG1-FITC, Becton-Dickinson, clone MOPC-21; IgG1-PE Becton-Dickinson, clone MOPC-21) at the same final concentration to set gate for positive events. AnxV-binding to MPs was measured using AnxV-APC (10 ng/mL final concentration, Becton-Dickinson) in the presence of 1 mM Ca2+. Platelet poor plasma aliquots (250 μL) were thawed on melting ice. Labeling of MPs for flow cytometric analysis was carried out by mixing 5 μL prediluted AnxV-APC and 5 μL prediluted specific antibodies or isotype-matched control antibodies with 5 μL heparin-sodium salt 10 % w/v (Sigma-Aldrich 194 USP/mg dry basis. Heparin was added to avoid clotting of the plasma). Finally, 5 μL PPP was added followed by dilution with 935 μL low phosphate buffered saline containing calcium, (PBS-Ca; 154 mmol/L NaCl, 1.4 mmol/L phosphate, pH 7.4, 2.5 mM CaCl2) and incubation for 1 hour in the dark. As a negative control experiment for AnxV binding, low phosphate buffered saline containing citrate (PBS-citrate; 154 mmol/L NaCl, 1.4 mmol/L phosphate, pH 7.4, 10.5 mM trisodium citrate) was used for dilution replacing the PBS-Ca.To reduce background noise, buffers were filtered through 0.1 μm pore size filters (MiniSart HF, Sartorius Stedim Biotech S.A., Aubagne, France). Fresh buffers were prepared on a weekly basis or more frequently if a rise in background noise was observed indicating contamination. The samples were analyzed using a FACS Calibur flow cytometer (BD Biosciences) controlled by CellQuest software version 5.1.1 in the “high” flow rate mode. Flow rate was measured before each experiment. Both forward scatter (FSC) and side scatter (SSC) were recorded with logarithmic gain. Acquisition time was 60 seconds. MP gating was accomplished using 1 μm beads (Flow Cytometry Size Calibration Kit, Molecular Probes, Inc., Eugene, OR, USA) for setting upper limits in both FSC and SSC signals, and a lower limit was placed to exclude buffer noise. The MP gate was validated by showing that the majority of AnxV+ MPs induced from platelets using the calcium ionophore A23187 (Sigma, Saint Louis, MO, USA) were detected using this gate (data not shown). MPs gated this way were further analyzed in SSC/FL-1, SSC/FL-2 or SSC/FL-4 plots to discriminate labeled particles from unlabeled particles using a fluorescence threshold determined by the fluorescence level of the MPs stained using the isotype-matched control antibodies and AnxV non-binding controls. Instrument settings were as previously described []. Flow cytometric data analysis was performed using FlowJo software version 7.6.1 (Tree Star, Inc., Ashland, OR, USA).MP-plasma concentrations of MPs were calculated as MPs/mL based on MP count per unit time, flow rate of the flow cytometer and net dilution during sample preparation of the analyzed sample. All samples were double-labeled. The total cell derived population was calculated as the sum of all the cell derived MP populations (EMPs, LMPs and PMPs). [...] Pearson’s correlation analysis was used to examine relationships between MP concentrations, lung function tests, and estimated pulmonary arterial pressure and clinical blood samples. Data were logarithmically transformed allowing parametric tests. In order to compare MP concentrations between patients with and without lung fibrosis Student’s t-tests were performed on logarithmically transformed data. Student’s t-test was used to compare the distribution of different clinical features in the different SSc patient subsets. Statistical significance was defined as p < 0.05. GraphPadPrism v. 5 and Microsoft Excel 2010 were used for the statistical calculations and plots. Data on MP concentrations and soluble selectin levels [] are here correlated with clinical parameters, […]

Pipeline specifications

Software tools FlowJo, GraphPad Prism
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Fibrosis, Hypertension, Pulmonary, Scleroderma, Systemic