Phycoerythrin (PE) conjugated IgG2a isotype control (IgG-IC) was obtained from Immunotech, (Marseille, France). Mab to human CD59 (PE-conjugated, clone MEM 43) was from Caltag Lab. (Burlingame, CA), Mab to human CD105 (PE-conjugated, clone N1-3A1) was from Ancell/Alexis (San Diego, CA).
Endothelial cell culture
HUVEC were obtained from Clonetics (San Diego, CA) and cultured in endothelial cell growth media EGM-2 (Clonetics) containing 2% of fetal bovine serum and supplements. Cells of 2nd passage were used in the experiments. Cells were seeded at a density of 10,000 cells/cm2 in polystyrene Falcon six-well plates or 60 mm dishes (Becton Dickinson Labware, Franklin Lakes, NJ) and maintained at 37°C under an atmosphere of 5% of CO2 and 95% room air. Cells were used for experiments at 48 hrs in culture when they reached confluence. For apoptotic assays, treated culture was washed with Hanks' balanced salt solution (HBSS) with 0.35% bovine serum albumin (HBSS/BSA) and adherent cells were harvested with 20 mM HEPES buffer with 100 mM NaCl, 0.5% BSA and 10 mM EDTA, pH 7.4. After harvest, cells were centrifuged (300 g/5 min), washed with HBSS/BSA and used for analysis.
Phase contrast microscopy
HUVEC cultures in 6-well plates were photographed using CK40 Olympus culture microscope (Olympus, Japan) with a SPOT camera and software (Diagnostic Instruments, Inc., MI, USA). Phase contrast at 200× magnification was used. The culture cell density was estimated by visual counting of cells in a standard area on the micrographs.
Overnight stimulation of HUVEC
Confluent HUVEC were washed with HBSS and incubated for 24 hrs at 37°C with various agonists in the medium: MEDIUM only; DMSO 0.5 % (vehicle for camptothecin); tumor necrosis factor-α (TNF) from Alexis Biochemicals (San Diego, CA) 100 or 1000 U/mL; cycloheximide (CHX) 50 μg/mL; TNF 1000 U/mL with cycloheximide 50 μg/mL (TNF/CHX); lipopolysaccharide (LPS) E. coli 055:B5 from Calbiochem (San Diego, CA) 10 μg/mL; camptothecin (CPT) from Sigma (St. Louis, MO) 5 μmol/L or 500 nmol/L; CPT with caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone (ZVAD) from Calbiochem (San Diego, CA) 50 μmol/L or ZVAD 50 μmol/L alone. After incubation, media and cells were harvested for analysis.
In a study of MP-release in subconfluent, confluent and overgrown HUVEC culture, the cells at 24 hrs (50% of confluence), 48 hrs (reaching confluence) or 72 hrs (overgrown) in culture were incubated for 24 hrs with 0.5 % DMSO, 500 nM or 5 μM CPT in medium. After incubation supernatant from cell culture was harvested and annexin V-FITC-binding MP were counted.
In a study of expression of CD59 and CD105 on endothelial cells, apoptotic bodies and other cell fragments, the confluent HUVEC at 48 hrs in culture were treated for 24 hrs with 0.5% DMSO, 500 nM or 5 μM CPT in medium. After treatment the cell culture supernatant was separated, adherent cells were harvested with 20 mM HEPES buffer with 100 mM NaCl, 0.5% BSA and 10 mM EDTA, pH 7.4, and pooled with culture supernatant. This mixture of cells, apoptotic bodies and other cell fragments was centrifuged (300 g/5 min), washed with HBSS/BSA, labeled in aliquots with saturating concentration of PE-conjugated Mab against CD105 or CD59, washed again in HBSS/BSA and analyzed by flow cytometry.
Short-time stimulation of HUVEC
Confluent HUVEC were washed with HBSS and incubated for 30 min at 37°C with various agonists in HBSS with 0.35%BSA: HBSS alone; 0.5 % dimethyl sulfoxide (DMSO); ionophore A23187 1–100 μmol/L; phorbol myristate acetate (PMA) 1 μg/mL; human thrombin receptor activating peptide SFLLRNP (TRAP) 1–50 μmol/L; human thrombin (THR) US FDA standard lot J (Bethesda, MD) 0.1–10 NIH Unit/mL; human tissue plasminogen activator (TPA) 3.5 kU/mL or human angiotensin II (AGTII) 1 μmol/L. All chemicals were from Calbiochem (San Diego, CA) unless otherwise stated. After incubation, cell cultures were observed by phase contrast microscopy and cell supernatants were collected for MP analysis.
Annexin V-labeling of microparticles
One hundred μL of medium or cell culture supernatant were incubated with 4 μL of annexin V-FITC from BD PharMingen (San Diego, CA) for 20 min at room temperature with or without 50 mM EDTA to estimate the non-specific binding. Fifty μL of labeled MP were added to TruCount tubes (Becton Dickinson) containing standard beads in 450 μL of HBSS/BSA solution. Samples were immediately analyzed with flow cytometry.
Immunolabeling of annexin V-binding microparticles
Medium or cell supernatant containing MP were centrifuged at 2,700 g for 5 min, and the sediment was discarded. Supernatant was further centrifuged at 19,800 g for 5 min at 10°C, and sedimented MP were resuspended in the original volume of HBSS/BSA. Aliquots of 50 μL were incubated for 20 min at room temperature with 2 μL of annexin V-FITC from BD Pharmingen (San Diego, CA) and saturating concentrations of PE-conjugated Mab. After incubation and washing with 1 mL HBSS/BSA (centrifuged at 19,800 g for 5 min at 10°C) samples were diluted with 450 μL HBSS/BSA and analyzed by flow cytometry.
Flow cytometry of endothelial cells and cell fragments
Cell samples were analyzed by a FACScan flow cytometer (Becton Dickinson, San Diego, CA) equipped with CELLQUEST software with forward scatter (FSC) and side scatter (SSC) in linear mode setting. Samples were analyzed using forward scatter (FSC) vs. side scatter (SSC) plot and propidium iodide (PI) fluorescence vs. FSC plot. Single cells were gated separately in gate G1 and apoptotic bodies and other cell fragment in gate G2 (Fig. 5). Count of 10,000 gated particles was analyzed for each sample. Apoptotic status of cells was assayed using Annexin V-FITC Apoptosis Detection Kit for annexin V/ propidium iodide cell labeling and using Apo-Direct Kit for terminal deoxynucleotidyltransferase dUTP-FITC nick end labeling (TUNEL) according to manufacturer's instruction (BD Pharmingen, San Diego, CA). Percentages of propidium iodide-negative cells binding annexin V and TUNEL-positive cells were evaluated. In a study of the expression of CD59 and CD105 on HUVEC, histograms showing expression of CD105 and CD59 on cells (G1) and cell fragments (G2) are presented. Binding of the IgG2a isotype control (IgG-IC) corresponded to the fluorescence of nonlabeled cells and cell fragments. Standard Quantum TM24 beads (Flow Cytometry Standards, San Juan, PR) were run each day as a separate sample to standardize fluorescence readings.
Flow cytometry analysis of endothelial cell membrane microparticles
MP were analyzed in a separate protocol  using a modified method of Combes et al. . Annexin V-FITC-positive MP were gated in a SSC vs. fluorescence logarithmic plot. For MP quantitation, TruCount beads from Becton Dickinson (San Diego, CA) were used in each sample as an internal standard. TruCount beads were gated in a SSC vs. FSC logarithmic plot and analysis was stopped when 5,000 beads were counted. MP count released by 103 cells was calculated for each sample. TetraSpeck fluorescent microsphere standards of 0.2, 0.5, 1.0 and 4.0 μm in diameter from Molecular Probes (Eugene, OR) were used to estimate MP size based on comparison of FSC values. Antigen expression on MP was analyzed by double-labeling with annexin V-FITC and PE-conjugated Mabs. The annexin V-FITC-positive MP were gated, and their binding of PE-labeled Mabs was evaluated in SSC vs. PE – fluorescence logarithmic plots.