Control mouse igg2a

Murine whole blood was drawn by cardiac puncture from Jcl:ICR or Crl:CD1 (ICR) mice terminally anesthetized with chloroform or sevoflurane into syringe containing sodium citrate at a final concentration of 0.38% or heparin at a final concentration of 10 units/ml (Mochida Pharmaceutical, Tokyo, Japan). Platelet-rich plasma (PRP) was collected from supernatants of murine whole blood by centrifugation at 110 g for 8 min. Washed platelets were prepared from PRP pellets by centrifugation at 500 g for 10 min, followed by a final wash with modified Tyrode’s solution (137 mM NaCl, 11.9 mM NaHCO₃, 0.4 mM Na₂HPO₄, 2.7 mM KCl, 1.1 mM MgCl₂, 5.6 mM glucose)52 (link). Washed platelets were resuspended in modified Tyrode’s solution containing 2% platelet-poor plasma (PPP) at a concentration of 2–3 × 10⁸ platelets/ml. Before experiments, 250 μM CaCl₂ was added to platelet suspensions. Platelet aggregation assay using a platelet aggregometer (MCM Hema Tracer 313 M; SSR Engineering, Kanagawa, Japan) was performed as previously described12 (link). Briefly, 5 × 10⁴ tumour cells in 10 μl PBS were added to the platelet suspension as platelet aggregation inducers. In some experiments, UM-UC-5 or SCC-015 cells were incubated with 1–100 μg/ml anti-podoplanin mAb (MS-1 or PG4D2) or control mouse IgG2a (Sigma Aldrich) for 30–45 min on ice prior to the platelet aggregation assay.

HEK 293T cells transfected with mouse or human NTPDase8, were detached from the plates with a citric saline solution (135 mM potassium chloride, 15 mM sodium citrate). Samples of 2.5 × 10⁵ cells per tube were washed with an ice-cold PBS solution containing 1% FBS and 0.1% NaN₃ [fluorescence-activated cell sorting (FACS) buffer] followed by incubation with the primary antibodies (serum from polyclonal anti-mouse NTPDase8 or purified monoclonal antibodies to human NTPDase8) or negative control (guinea pig preimmune sera or control mouse IgG2a (Sigma-Aldrich, Oakville, ON, Canada)) in FACS buffer for 1 h. After washes with FACS buffer solution, the cells were incubated with an appropriate Alexa-conjugated secondary antibody for 30 min on ice, washed with FACS buffer, and analyzed by flow cytometry (BD LSR II, BD Biosciences, San Jose, CA USA).

Cells were plated on chamber slides coated with 10 μg/ml fibronectin (Sigma-Aldrich, St. Louis, MO), then treated as above and subjected to immunofluorescence as previously described using FITC-conjugated mouse monoclonal anti-α-smooth muscle actin (αSMA) (Sigma-Aldrich, St. Louis, MO), and biotin conjugated rabbit polyclonal anti-collagen type 1 (COL1) (Rockland Immunochemicals, Gilbertsville, PA), PE-Cy 5 streptavidin (BD Pharmingen San Diego, CA) secondary antibodies, and control mouse IgG2a (Sigma-Aldrich, St. Louis, MO), and rabbit IgG biotin conjugate (Rockland Immunochemicals, Gilbertsville, PA) [14 (link)]. Photomicrographs were taken on an Olympus BX53 fluorescence microscope.