The percentage of annexin A5 single-positive

cells (early apoptotic cells) was calculated within the viable population of cells. Enumeration of hypoploid cells was carried out as described previously [25, 26]. Briefly, cell pellets were resuspended and fixed with 70% ethanol for 2 h at −20°C. Subsequently, cells were centrifuged and resuspended in PI incubation buffer (45 mM Na2HPO4, 2·5 mM citric acid and 0·1% Triton X-100) for 20 min at 37°C. PI was added to a final concentration of 10 μg/ml. All cell preparations were examined with a FACSCanto II (BD Biosciences) using the diva software 3-MA concentration (BD Biosciences) for analysis. Doublets were ‘gated-out’ by making use of a two-parameter measurement scheme in which a plot of

pulse peak height versus area (integral) PI signal allowed for identification and exclusion of doublets. The principles and components this website of RT–CES™ (ACEA Biosciences Inc., San Diego, CA, USA) technology have been described previously [27-29]. Briefly, the RT–CES system allows for non-invasive monitoring of target cells by using impedance sensor technology. Electrode impedance, which is displayed and recorded as cell index (CI) values, reflect the biological status of monitored cells, including the cell number, cell viability, morphology and adhesion quality. We monitored the effects of purified IgG from a subgroup of PAH (n = 16), SSc (n = 12) and SLE nephritis (n = 6) patients and healthy controls (n = 6) on HUVECs with the RT–CES™ system. We performed three experiments with the RT–CES™ system, each experiment with different HUVEC batches but with the same purified IgG from the above-mentioned subgroups. HUVECs were seeded at a density of 4500 cells per well on 96-well plates integrated with microelectrodes at the bottom of the wells (E-plates™; ACEA Biosciences Inc.). Briefly, cells were trypsinized, centrifuged and resuspended Histone demethylase in culture medium consisting of RPMI-1640 with Glutamax-1 (Gibco) supplemented with 10% iFCS (Integro BV) and counted. Background measurements were

taken after adding 50 μl of the culture medium to the wells of the E-Plate™. Cells were adjusted to the appropriate concentration, and 100 μl of the cell suspension was added to the E-plate™ wells. Thereafter, cell attachment, spreading and proliferation were monitored every 15 min using the RT–CES system. The cells were in the log growth phase after approximately 2–3 h after seeding, depending on the HUVEC batch used in the respective experiment. At this point, being similar within each HUVEC batch, the cells were treated with 160 μg/ml patient or control IgG in triplicate and monitored continuously for 48 h. HUVECs incubated in culture medium without iFCS (cell starvation) and HUVEC treated with 5 nmol/ml staurosporine in 10% iFCS served as internal positive controls for apoptosis. Data were analysed with spss statistical software version 15·0 for Windows.