Amg inverted phase contrast microscope

Wound scratch migration assays were performed as previously described [25 ]. Briefly, 2×10⁵ cells per well were seeded in a 24-well plate. A scratch wound was applied on confluent ECs monolayer using a 10 μL pipette tip, and then photos were taken as T0 using an AMG inverted phase-contrast microscope (AMG, Life Technologies, NY). The cultures were further incubated for 12 hours and photographed again (T12). Migration distance (the average of three measurements of gap distance at T0 minus the average of three measurements of gap distances at T12 and then divided by two) was measured with NIH Image J software and is expressed in μm.

Tube formation assay was performed as previously described [25 (link)]. Briefly, ECs (2 × 10⁴ cells/well) were seeded into a 96-well plate pre-coated with 40 μL reduced-growth factor ECM gel (Life Technologies, NY, USA) and incubated in a regular incubator or in an air-tight chamber (MIC-101, Billups-Rothenberg, Inc., CA, USA) flushed with 95% N₂/5% CO₂, at 37 °C for 6 hours. Photos of tube-like structure were captured with an AMG inverted phase-contrast microscope (AMG, Life Technologies, NY, USA). The numbers of branching points and segments were quantified by NIH Image J software with angiogenesis analyzer plug-in (developed by Gilles Carpentier, http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzer-for-ImageJ).