Imagexpress micro widefield platereader

HUVECs (passage 4-9) were seeded at 5,000 cells/well in black flat-bottomed 96-well plates (Greiner Bio-One, 655090) in 10% LVES/Medium 200. Following 24 hours of cell growth at 37°C/5% CO₂, plating medium was replaced with Medium 200 containing 0.1% serum for 24 hours. Cells were then stimulated with commercially available VEGF₁₂₁a or VEGF₁₆₅b (R&D Systems), VEGF₁₂₁a-TMR or VEGF₁₆₅b-TMR (Promega Corporation, USA) at 0.3nM, 3nM or 30nM (in 0.1% serum/medium), or positive control 3nM VEGF₁₆₅a (R&D Systems). Following 48 hour stimulation at 37°C/5% CO₂, cells were washed with 100μl/well PBS, fixed with 3% PFA/PBS (20 minutes, room temperature ) and nuclei stained with 2mg/ml H33342 (15 minutes, RT). Nuclei were imaged using an ImageXpress Micro widefield platereader (Molecular Devices, USA) with a 4x objective using a DAPI filter (4 sites per well, 25ms exposure time).

Images obtained with the ImageXpress Micro widefield platereader at 4 sites per well were quantified using MetaXpress 2.0 (Molecular Devices, USA). Nuclei were quantified with diameter 5-25μm and 100 graylevel intensity above background. VEGFR2 phosphorylation was quantified (Figures 1D and 1E) using a granularity algorithm, granules were defined as 6-12μm diameter with a graylevel intensity of 50 above background. Granularity was quantified per cell, baseline-corrected to non-specific binding (secondary antibody only) and normalised to cediranib-treated wells (0%) and response to 30nM VEGF₁₆₅a (100%). Quantifying relative receptor expression (Figure S4) using a multiwavelength cell scoring algorithm, regions were defined as 2-15μm in size. Due to distinctions in secondary antibodies, VEGFR2 (FITC) was defined as intensity over 200 graylevels and NRP1 (TRITC) over 50 graylevels. Fluorescence was quantified as integrated intensity per cell and baseline-corrected per experiment to non-specific fluorescence (secondary antibody only).