Qwin lite software

Fertilized white Leghorn eggs were incubated at 37°C in a humidified incubator and windowed. On day 7 of development, sterile filters soaked with either vehicle or VEGF (100 ng/disk) in the presence or absence of NC (9 µg/disk) were applied to relatively avascular regions of the CAM. CAMs were fixed (4% paraformaldehyde in PBS) in ovo on day 9 and photographed in the localized area of the filter. The newly capillarized area in the region of each filter was quantitated using Leica QWin Lite software and neovascularization is expressed as an angiogenic index (n= 12–15 eggs per treatment). The present study was approved by the Ethics Committee of The First Affiliated Hospital of Wenzhou Medical University.

HUVECs were plated into 24-well plates (1 × 10⁵cells/well) in EGM containing 10% FCS and confluent monolayers were starved for 24 h by replacing medium with EGM containing 0,5% FCS. HUVECs were then co-cultured with Jurkat cells previously nucleofected or not with AG49CMVGag-RTEm26CTE (Jurkat-Gag) and pEGFPN3 (Jurkat-EGFP). After 24 h of culture, Jurkat cells were removed and HUVEC monolayers were scratched using a 200 μL pipette tip. In some experiments starved HUVEC monolayers were scratched, washed with warm PBS and cultured in EGM containing 10% FCS in the presence or absence of synthetic p17s at different concentrations. HUVEC migration was evaluated at different time points using an inverted microscope (DM-IRB microscope system, Leica, Milan Italy). HUVECs migrating into the wounded area, or protruding from the border of the wound, were photographed using a CCD camera (Hitachi Inc., Krefeld, Germany) connected to a computer via a frame grabber (Matrox Meteor). Analysis of the images were performed using the QWin-lite software (Leica).

The wound healing assay was performed following previously described procedures with minor modifications [14 (link)]. Cells (1 × 10⁵) were plated into collagen-coated 24-well plates overnight. Twenty-four hours later, the monolayer was scratched using a 200 μL pipette tip and cultured in complete medium. The percentage of wound healing was evaluated during a period of 8–10 h. ECs migration was recorded using a DM-IRB microscope system (Leica, Wetzlar, Germany), equipped with a Charge Coupled Device (CCD) camera (Hitachi Ltd., Tokyo, Japan) and connected to a computer via a frame grabber (Matrox Meteor). Analysis of the images was performed using the QWin-lite software (Leica). In some experiments, confluent EC monolayers were pretreated, before scratching, with conditioned medium from Mock- or ARV p17-nucleofected cells for 16 h. In other experiments, EC monolayers were scratched and then cultured in the presence of 10 μM Diprotin A (DPA; Abcam, Cambridge, UK).