Trypsin solution

After the treatment of SKOV-3 cells (24 or 48 h) with ten different ascites samples and the control condition (culture medium), cell cultures were washed with sterile 1 × PBS and were subsequently recovered using trypsin solution (Corning, 25-051-CI). Cell lysis was carried out using RIPA buffer (5 mM Tris–HCl, 2 mM EDTA, 50 mM NaCl, and 1% Nonidet P-40) with a cocktail of protease and phosphatase inhibitors (1 mg/ml aprotinin and leupeptin and 1 mM PMSF, NaF, and Na₃VO₄). Finally, the samples were quantified using a DC Protein Assay Kit (Bio-Rad, 500-0114).

Human umbilical vein endothelial cells (ATCC, Virginia, USA; CRL-2873) were used for this in vitro model of hypertensive disorders of pregnancy. Cells were cultivated in sterile 25 cm
²flasks using growth medium (Gibco, CA, USA) supplemented with fetal bovine serum 10% v/v (Gibco), 50 μg/ml penicillin, 50 μg/ml streptomycin, and 0.5 μg/ml amphotericin B (Gibco). For the experiments described ahead, cells were detached from culture flask (Corning, Costar, Netherlands) using trypsin solution (Trypsin/EDTA 0.5/0.2 mg/ml in phosphate-buffered saline, PBS) centrifuged at 1,200 rpm for 10 minutes, resuspended in growth medium free from fetal bovine serum containing antibiotic and antifungal solution and seeded on 96 well microplate (1.10
4 (link)
cells/well) overnight at 37°C, 5% CO
₂tension, and 95% humidity to ensure cell adhesion
_.

The following breast cancer cell lines, namely triple negative breast cancer (TNBC) HCC1806 (CRL-2335^™), ER-positive HCC1428 (CRL-2327^™), and HER2-positive AU565 (CRL-2351^™) (ATCC, Manassas, VA, USA), were grown in RPMI-1640 medium (Corning, Tewksbury, MA, USA) supplemented with 10% FBS and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 g/mL amphotericin). MCF10F non-tumorigenic epithelial cells from mammary gland (CRL-10318^™, ATCC, Manassas, VA, USA) were cultivated in DMEM/F12 medium (Merck KGaA, Darmstadt, Germany) containing 5% horse serum, 10 ng/mL epidermal growth factor EGF, 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, 100 ng/mL cholera toxin, and mix of antibiotics. Cells were cultured at 37 °C in a controlled humidified atmosphere containing 5% CO₂ and passaged with trypsin solution (0.05% trypsin for MCF10F cells and 0.25% trypsin for breast cancer cells, respectively, Corning, Tewksbury, MA, USA).

Cardiac fibroblasts were cultured on petri dishes as previously described. At passage 5, 5 plates of cells were cleaved with 0.25% trypsin solution (Corning). Cells were then spun down into a pellet and resuspended in 1% BSA/PBS at a concentration of 1000 cells/μL. Cells were then placed on ice for ~ 45 min before targeting 5000 cells on the Chromium Controller instrument using the Chromium Next GEM Single Cell 3′ reagent Kit, v3.1 (10× Genomics) according to the manufacturers protocol. Single cell partitioning, cDNA library preparation, and sequencing using a NovaSeq6000 was performed by the University of Texas Genomic Sequencing and Analysis Facility. An Agilent Bioanalyzer (Agilent) and the KAPA SYBR FAST qPCR kit (Roche) were used to determine the quality and concentration of the finished library. Libraries were sequenced to a depth of 50,000 reads per cell.

HUVEC cells were cultured to confluency in FN1-coated 6-well plates. The cells were then serum-starved for 2 h with 100 µM AXT107 or equivalent DMSO added after 30 min. TNFα (10 ng/mL) was then administered for 0, 2, 4, or 24 h. The addition of TNFα was staggered so that all samples could be harvested at the same time. The cells were then washed twice with dPBS without Ca²⁺ and Mg²⁺, detached with 0.025% Trypsin solution (Corning), and neutralized with Trypsin Neutralizing Solution (Lifeline Cell Technology). Cells were then washed once in dPBS without Ca²⁺ and Mg²⁺, resuspended in stain buffer (PBS, 4% FBS, and 0.09% sodium azide), and transferred into flow tubes at 1 × 10⁵ cells per tube. The cells were then washed twice in stain buffer, decanted, and then stained with 5 µL of PE-conjugated primary antibodies for VCAM-1 and ICAM-1 added directly to the remaining volume (approximately 100 µL). After 40 min, the cells were washed three times with stain buffer. During these last washes, a tube of Quantibrite^TM PE Quantitation beads (BD Biosciences) was also prepared. Data were collected using the FACScalibur flow cytometer and FACS Diva software (BD Biosciences) and analyzed with FlowJo software (BD Biosciences). The number of molecules per cell was quantified from geometric means using the Quantibrite^TM bead standards (BD Biosciences) according to the manufacturer’s protocol.