Trypsin

HT1080 cells derived from a human fibrosarcoma were obtained from the European Collection of Animal Cell Cultures, Porton Down, UK. HT1080s were maintained in a humidified incubator with 5 % CO₂ at 37 °C in DMEM (Gibco), containing 10 % fetal bovine serum (Gibco) and 1 % streptavidin/penicillin (Life Technologies). Prior to cell adhesion/spreading experiments, HT1080 s were detached from the cell culture flasks with 0.05 % trypsin/0.02 % EDTA (GE Healthcare), washed and re-suspended in serum free DMEM. Platelets were obtained from human platelet rich plasma provided by the National Health Service Blood and Transplants authority in accordance with the Declaration of Helsinki. Platelets were prepared by centrifugation for 15 min, 240 g, the pellet discarded and 1 μl of Prostaglandin E₁ (100 μg/ml in ethanol) added per ml of platelet supernatant. The platelet suspension was centrifuged at 640 g for 10 min and the platelet pellet resuspended in tyrodes buffer (140 mM NaCl, 5.6 mM Glucose, 2 mM MgCl₂, 0.4 mM NaH₂PO₄, 12 mM NaHCO₃, 2.7 mM KCl, 10 mM HEPES pH 7.4) to a density of 1 × 10⁸/ml.

Human fibrosarcoma cells, HT1080, were obtained from the European Collection of Animal Cell Cultures (Porton Down, UK). Rugli cells, derived from a rat glioma, were a kind gift from Dr. J. Gavrilovic, University of East Anglia, UK. Both cell lines were maintained at 37 °C in Dulbecco's modified Eagle's medium (DMEM, Sigma–Aldrich) containing 10% (v/v) foetal bovine serum (Sigma–Aldrich) and 1% (v/v) PenStrep (Life Technologies). Prior to cell binding or cell spreading experiments, cells were detached from cell culture flasks at a confluence of about 75% with 0.05% trypsin/0.02% EDTA (GE Healthcare), washed and resuspended in serum free DMEM.

The Vero (African green monkey kidney; ATCC CCL-81) cell line was maintained in Dulbecco modified Eagle medium (DMEM; Corning), supplemented with 10% fetal bovine serum (FBS; Peak Serum), 1% penicillin and streptomycin (Pen-Strep; Corning), HEPES (Gibco), and 0.1% amphotericin B (Geminin Bio-Products). The JAR (Human Placental; ATCC HTB-144) cell line was maintained in RPMI medium (Corning) supplemented with 10% FBS and 1% Pen-Strep. Vertebrate cell lines were maintained at 37°C with 5% CO₂ and propagated to new flasks using 0.05% trypsin (GE Healthcare Life Sciences). The C636 (A. albopictus; ATCC CRL-1660) cell line was maintained in MEM (GE Healthcare Life Sciences) with 10% FBS and 1% Pen-Strep. The Aag2 (A. aegypti; RRID:CVCL_Z617) cell line was maintained in Schneider’s insect medium with 10% FBS and 1% Pen-Strep. Both insect cell lines were maintained at 28°C, C636 with 5% CO₂, Aag2 in sealed flasks, and propagated to new flasks by cell scraping.