Axio observer

For images in 2D, 1 × 10⁵ tumor cells were plated on 12 well plates and the day after treated with either 100 μg/ml LDL or the same volume of control (150 mM NaCl, 0.01% EDTA) for 6 h. After that, cells were trypsinized and 0.5 × 10⁴ cells were plated on top of confluent monolayers of HUVECs, that had been plated the day before at 2 × 10⁴ cells per well on top of 0.2% gelatin (8 well 1 cm² area ibidi plates). Cells were imaged on a Zeiss Axio Observer under controlled CO₂ and temperature. Pictures were taken every 5 min at both transmitted (green channel) and reflected light using a Zeiss Axio Observer under controlled CO2 and temperature.

In vitro flow studies were performed on multichannel microfluidic capillary systems which had been coated with either 300 µg/ml fibrinogen or 25 μg/ml collagen overnight at 4 °C and then blocked with 5% BSA (5 mg/ml) for 1 hour. Whole blood from healthy donors was obtained in PPACK (50 µM) and platelets were fluorescently labelled with 3,3′-Dihexyloxacarbocyanine Iodide (10 µM) prior to being flowed for 2 minutes at a shear rate of 1000 s⁻¹ (37 °C). Following this initial flow to adhere platelets and form aggregates, buffer only or GSNO (100 nM) were post-flowed over the adhered platelets for a further 20 minutes prior to fixation with 4% paraformaldehyde. Fixed adhered platelets were either restained overnight with 3,3′-Dihexyloxacarbocyanine Iodide (10 µM) or permabilised in 0.1% Triton X-100 and stained with FITC-phalloidin before imaging using the Zeiss Axio Observer (x63 oil immersion objective, 1.4 NA (Zeiss, Cambridge, UK)). Acquired images were analysed using ImageJ (NIH, Bethesda, USA). Thrombus height was calculated by imaging the thrombi using the Apotome 2 confocal unit on the Zeiss Axio Observer (x63 oil immersion objective, 1.4 NA (Zeiss, Cambridge, UK)). Images were taken at 0.5 μm depth from the top of the thrombus to the bottom. All thrombi in a field of view were identified and the total height of the thrombi identified.