Ebm singlequots

Human SW480 and SW620 colon adenocarcinoma cells derived from primary and secondary tumours resected from a single patient, respectively, were obtained from ATCC (Manassas, VA, USA). Low-metastatic NM11 and high-metastatic LuM1 cells were established from the murine colon adenocarcinoma Colon 26 cell line50 (link) and supplied by Dr S. Shimizu, Aichi Cancer Center, Nagoya, Japan These cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). HUVECs were purchased from Lonza Walkersville, Inc. (Walkersville, MD, USA) and grown in endothelial basal medium (EBM) supplemented with EBM SingleQuots (Lonza), FBS, hEGF, hydrocortisone, GA-1000 and BBE. The murine macrophage cell line RAW264.7 was kindly supplied by Dr M. Nakamura, Collaboration Center, Shimane University, Izumo, Japan. These cells were maintained at 37 °C under 5% CO₂. ExpiCHO-S cells were purchased from Gibco, Thermo Fisher Scientific Inc. (Waltham, MA, USA) and cultured in ExpiCHO expression medium at 37 °C under 8% CO₂ with shaking in an Erlenmeyer flask. All the cell lines were not authenticated after purchase or transferred from other laboratories (propagated and stored in liquid N₂ after receipt). All cell lines were free of mycoplasma contamination as assessed by e-Myco Mycoplasma PCR Detection Kit (Cosmo Bio Co Ltd., Tokyo, Japan).

Immortalized HUVECtert cells (Schiller et al., 2009 (link)) were maintained in EBM™ growth medium supplemented with 10% FBS, EBM SingleQuots (Lonza, Basel, Switzerland), 100 U·mL⁻¹ benzylpenicillin, 100 μg·mL⁻¹ streptomycin and 1% amphotericin. The HUVECtert cell line was used instead of primary HUVEC in order to overcome batch-to-batch variability. The cells were seeded at a density of 5 × 10⁵ cells per well overnight in 6-well plates. Then, cells were pretreated with plumericin for 30 min before TNF-α (10 ng·mL⁻¹) stimulation for additional 14 h (VCAM-1 and ICAM-1 quantification) or 5 h (E-selectin). Cells were stained with FITC-labelled antibodies [anti-VCAM-1 (BD Biosciences, Schwechat, Austria; 551146), anti-ICAM-1 (eBioscience, Vienna, Austria; BMS108FI), anti-E-selectin (Bio-Rad AbD Serotec, Puchheim, Germany; MCA1969F)] and analysed with a FACSCalibur™ (BD Biosciences) flow cytometer.