Inverted contrast phase light microscope

Six 8- to 10-week-old male Sprague-Dawley rats were purchased from National Laboratory Animal Center (Taipei, Taiwan) and used for the aortic ring sprouting assay. Rats were sacrificed using CO₂ asphyxiation to dissect the aortic arches. The surrounding fibro-adipose tissues were removed. The aortas were thoroughly rinsed with M199 medium and cut into approximately 1 mm ring segments. In each experiment, the aortic rings obtained from one rat were utilized for different treatment groups. The aortic rings were immersed in Matrigel in the wells of a 48-well tissue culture plate. VEGF-A (25 ng/ml) with or without MFB was added to the wells. The aortic rings were cultured in a humidified 37°C incubator and the cultured medium was changed every 3 days. Growing sprouts of endothelial cells were photographed under an inverted contrast phase light microscope (×40, Nikon, Japan) on day 7. The sprouting area was determined on the computer-digitized images with Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA) (Image-Pro Plus). An observer who was unaware of the treatment group assessed the sprouting area.

To perform the cell invasion assays, Transwell plates (Corning, NY, USA) were used as previously described [31 (link)]. Briefly, the bottom face of the insert membrane was covered with 0.2% gelatin. HUVECs in 200 µL 2%-FBS-containing M199 medium (10⁴ cells/well) in the presence or absence of PPE8 were seeded in the top chambers. The bottom chambers were filled with M199 medium containing 2% FBS with or without 25 ng/mL VEGF-A. After 18 h treatment, non-invaded cells were scraped with a cotton swab. Invaded cells (on the bottom side of the insert membrane) were fixed for 30 min with paraformaldehyde (4%) and stained with toluidine blue O (0.5%). An inverted contrast-phase light microscope (Nikon, Japan) was used to photograph the invaded cells at 40× magnification. The extent of cell invasion was determined by counting the invaded cells in three random fields.

Six male Sprague-Dawley rats (8- to 10-week-old) from the National Laboratory Animal Center (Taipei, Taiwan) were used for the aortic ring sprouting assay as previously described [31 (link)]. Rats were sacrificed using CO₂ asphyxiation to dissect the aortic arches. The surrounding fibro-adipose tissues were removed. The aortas were rinsed thoroughly with M199 medium and cut into approximately 1 mm ring segments. In each experiment, the aortic rings obtained from one rat were utilized for different treatment groups. The aortic rings were immersed in Matrigel and treated with VEGF-A (25 ng/mL) in the presence or absence of PPE8. The treated aortic rings were placed in a humidified 37 °C incubator and the medium was changed every 3 days. An inverted contrast-phase light microscope (Nikon, Japan) was used to photograph the growing sprouts of endothelial cells at 40× magnification on day 7. The sprouting area was determined by an observer who was unaware of the treatment group. Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA) (Image-Pro Plus) was employed to quantify the sprouting area.