μ slide angiogenesis wells

Growth factor reduced Matrigel™ (BD Discovery Labware, Bedford, MA, USA) was placed into the lower chambers of μ-slide angiogenesis wells (IBIDI, Planegg/Martinsried, Germany) and hardened for 30 min at 37°C. Then, 1.5×10⁴ LECs/wells were seeded on the Matrigel and were either left untreated (solvent only, Ethanol) or incubated with TSA (at 400 nM for 6 h). Images were taken on the Biozero BZ-8000K microscope (Keyence Deutschland GmbH, Frankfurt am Main, Germany). Image acquisition was performed using BZ analyser software (Keyence Deutschland GmbH, Frankfurt am Main, Germany).

Tube formation assays were performed in μ-Slide Angiogenesis wells (ibidi GmbH, Germany) using 10 μl of growth factor-reduced Matrigel (BD Bioscience) per well and allowed to polymerize for 30 min at 37 °C. Subsequently, 1 × 10⁴ HBMECs were resuspended in control CM, control CM+50 ng ml⁻¹ VEGF, MN-CM or IN-CM (all with and without adding 1 μg ml⁻¹ recombinant mouse Flt1/Fc), and 50 μl of cell suspension were added per well. Cells were incubated for 4–6 h in a humidified chamber at 37 °C, 5% CO₂. Images were acquired using a microscope Zeiss Axiovert 200 M with 5x/0,16 EC Plan-NEOFLUAR Objective. Tube length was quantified using Angiogenesis Analyzer for ImageJ (Gilles Carpentier. ImageJ contribution: angiogenesis analyzer. ImageJ News, 5 October 2012) and number of branches were counted manually. Quantification was done blind to the experimental condition.