Phenol red free matrigel

Explant cultures were performed as previously described (34 (link), 40 (link)). Briefly, villous explants with potential EVT columns were carefully dissected and positioned on Transwell inserts (Millipore, Billerica, MA) pre-coated with 200 μL of undiluted phenol red-free Matrigel (BD Biosciences, Bedford, MA). Explants were left overnight to attach to the Matrigel, at 37°C with 3% O₂ and 5% CO₂, before adding serum-free DMEM-F12 medium supplemented with 100 U/mL of penicillin, 100 U/mL of streptomycin, 100 μg/mL Normacin™. After 2 days of culture, villous tips were examined under the dissecting microscope for successful EVT outgrowths. All successful explants were selected for treatment with 200 nM oligomers (Table 3). Explants were photographed immediately after adding treatment, and subsequently at 48 h, using a Leica DFC400 camera attached to a dissecting microscope. ImageJ was used to measure the area of EVT outgrowth. Specifically, total outgrowth area was calculated by subtracting the area at the end point with the initial area upon treatment. Each experiment was designed with a minimum of four replicates, and was repeated on three different placentas.

Serum-free conditioned media (SFCM) were collected from 48 hrs cultures and centrifuges to remove cells. SFCM was then mixed with phenol-red-free matrigel (2:3 proportion, total 0.5 ml; BD Biosciences). Subcutaneous (s.c.) injection in mice was performed and the mice were killed after 8 days and the matrigel plugs were extracted for haemoglobin content (QuantiChromhaemoglobin assay; BioAssay Systems), and immunohistochemistry for CD31 antibody.

Animal experiments were performed according to protocols approved by the Care of Experimental Animals Committee (IACUC No. 2018-0018, approved date—18 June 2018). To induce xenografts, 1 × 10⁷ MDA-MB-231 or PC3 cells in phenol red-free Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were injected subcutaneously into the right flank of 6-week-old female or male Balb/c nude mice (Charles River Laboratories Japan, Inc., Yokohama, Japan) and grown for 2 weeks. For biodistribution analysis, 1.85 MBq Zr-89 ATPS mAb was administered intravenously (n = 5 for each time point), and then at 4, 24, and 48 h after injection, the mice were anesthetized, sacrificed, and dissected. The amount of radioactivity in the blood, heart, lungs, liver, spleen, stomach, kidneys, intestine, muscle, and tumor was measured using a gamma counter and the percentage of the injected dose per gram tissue was calculated. For the inhibition study (n = 3), 60 μg cold ATPS mAb was co-injected with 1.85 MBq (2.4 μg) Zr-89 ATPS mAb in mice bearing MDA-MB-231 tumors and then organs were removed at 24 h after injection. As a control, wild-type mice were injected with 1.85 MBq Zr-89 oxalate, an unlabeled form of Zr-89, and then organs were removed at 24 h after injection.