Basement membrane matrix growth factor reduced

The ability of LECs to form connections to nearby cells was analyzed with a matrigel tubule formation assay using a modification of a previously described protocol [17 (link)]. Briefly, cells were re-suspended in growth factor-reduced basement membrane matrix (BD Biosciences, San Jose, CA) at a concentration of 1.4×10⁵ cells per 100 μL of matrigel. After thorough mixing, 300 μL of the suspension was added to each well of a 24-well tissue culture plate. The plate was incubated at 37°C for 30 minutes to solidify the matrigel followed by addition of growth media containing 50 ng/ml of rhIL-4, rhIL-13, or media alone (control). Plates were cultured at 37°C and 5% CO₂ for 12 hours and then imaged using an inverted microscope (Carl Zeiss, Oberkochen, Germany) equipped with MetaMorph scanning and tubule analysis software (Molecular Devices, Sunnyvale, CA).

The EPCs derived from bone marrow of Sprague-Dawley (SD) rats were isolated and identified according to previous studies 34 (link),38 (link). The EPCs viability and proliferation was investigated through cell counting Kit-8 (CCK-8, Dojindo Co.) and Cell-Light™ EdU Apollo®567 In Vitro Kit (RiboBio Co., China) respectively. For tube formation assay, the cell suspension (5×10³ cells per well) pre-treated with PABC scaffold for 48 h was added into a µ-Slide (IBIDI, Germany) pre-coated with growth factor reduced basement membrane matrix (BD, Corning, US). After 6 h of incubation, the formed tubes were observed under a Nikon inverted light microscope. The number of tubes was counted according to the manufacturer's instructions. The particular steps and methods were described from the added files (supporting file).

Chick embryo metastasis assays were performed according to the method described by Zijlstra and colleagues 18 (link). Fertilized white leghorn chicken eggs (Goode Enterprises, Papaikou, HI) were incubated at 37°C with 70% humidity in a rotary incubator (1502 Sportsman; GQ Manufacturing, Savannah, GA) for 10 days. After this, the chorioallantoic membrane (CAM) underneath the eggshell was dropped by drilling a small hole in the air sac and a second close to the allantoic vein, not penetrating the membranes. A small incision was made into the eggshell membrane, leaving the CAM intact. A weak vacuum was used to drop the CAM and a 1 cm² opening was cut in proximity to the second incision near the allantoic vein. The CAM was abraded with a sterile cotton swab to access the mesenchyme. Cells were suspended in 50% DMEM/50% matrigel (Basement Membrane Matrix, Growth Factor Reduced, BD Biosciences, San Jose, CA). A 25-μL inoculum (containing 3 × 10⁶ cells) was pipetted onto the CAM and the hole was sealed with tape. The eggs were returned to a stationary incubator (1550 Hatcher; GQ Manufacturing) for an additional 7 days. After this incubation, the extra-embryonic tumor was excised and weighed. A sample of the lower CAM and the embryonic liver were harvested and analyzed for the presence of tumor cells by Alu PCR as described below.