Drabkin s reagent kit 525

The DIVAA was performed as described previously [38 (link)]. A subcutaneous pocket (5 × 10 mm) was created with a sterile hemostat in male C57BL/6 mice. Sterile silicone tubes were filled with Matrigel containing VEGF (200 ng/mL) in the presence or absence of 4AAQ, and these tubes were implanted into the dorsal flanks of the mice (each group contained six tubes in two independent experiments). Bevacizumab was used as positive control. The neovessel formation rate in these tubes was quantified by determining the hemoglobin levels in the Matrigel using a Drabkin’s Reagent Kit 525 (Sigma-Aldrich) after 15 days.

Six to eight week-old non-obese diabetic (NOD) severe combined immunodeficiency (SCID) NOD.CB17-Prkdcscid/NCrHsd mice (Envigo, Huntingdon, UK) were subcutaneously injected with a mixture containing growth factors reduced Matrigel and heparin (50 U/mL) supplemented with HGF and VEGF (200 ng/mL for each growth factor) or with RPMI8266 cell (5x10⁵ cells/mL) CM. Assuming an effect-size of 0.4 with statistical significance of α < 0.05 and a power of 80% we used n. 6 mice for each group for a total of 64 mice. This number was increased to 72 taking into account an expected drop-out rate of 10% for the treatment group. Drop-outs, specifically, may be caused by failure of plug injection. Mice were administered intraperitoneally every 72 hours with MP0250 (4 mg/Kg) or with PBS. After 14 days, mice were sacrificed, and the Matrigel plugs removed, weighed, and homogenized in cold PBS. The hemoglobin content of the plugs was calculated using Drabkin’s reagent kit 525 (Sigma-Aldrich) and normalized to the weights. Mice were housed according to the Institutional Animal Care and Use Committee of the University of Bari Medical School, Bari, Italy (licence n. 548/2016PR).

To assess the antiangiogenic effects of KC21 peptides in vivo, growth factor-reduced liquid Matrigel (0.5 mL) containing heparin (60 U/mL), VEGF (10 ng/mL, with the exception of control), and KC21 peptides or scramble peptides were subcutaneously injected into the mice near the abdominal midline. Seven days after injection, mice were euthanized and Matrigel plugs were surgically removed. For macroscopic analysis of angiogenesis, hemoglobin content in Matrigel was measured with Drabkin’s reagent kit 525 (Sigma-Aldrich).

Six-week-old male C57BL/6 and Redd1^−/− mice were injected with or without DOX (0.2 mg/kg/2 days, intraperitoneally). Matrigel (0.5 mL) containing 100 ng VEGF-A (or VEGF-C) and 10 U heparin was subcutaneously injected into mice 2 days after initial treatment with DOX under pentobarbital anesthesia (50 mg/kg, intraperitoneally). After 10 days, the mice were sacrificed by cervical dislocation, and the Matrigel plugs were carefully removed and photographed. Hemoglobin levels in the Matrigel plugs were measured using Drabkin’s Reagent Kit 525 (Sigma-Aldrich) to assess functional blood vessel formation. For identification of infiltrating vascular and lymphatic endothelial cells, immunohistochemistry was performed on slices of the Matrigel plug as described below.

The Matrigel plug assay was performed as described previously [24 (link)]. Briefly, growth-factor-reduced Matrigel (0.5 mL) containing 32 U heparin and 250 ng of mouse VEGF or mouse VEGF plus anti-PTK7 mAbs (3 and 10 μg/mL) was subcutaneously injected into female C57BL/6 mice aged 4 weeks. After 12 days, the mice were sacrificed and the plugs were recovered. The hemoglobin (Hb) content in the plugs was measured using Drabkin’s reagent kit 525 (Sigma-Aldrich) to quantify blood vessel formation.