Ls 3801

Measurement of ³⁶CI^– uptake was performed in accordance with a previously described method [15 (link)]. Briefly, 6 × 10⁶/ml neutrophils were incubated in HEPES buffer at 37°C for 1 h (in mM: 20 HEPES, 118 NaCl, 4.7 KCl, 1.2 MgSO₄ and 2.5 CaCl₂, pH 7.4). Neutrophils pretreated with phorbol myristate acetate (PMA) (2 μg/ml) for 15 min were exposed to 2 μCi/ml ³⁶CI^– in the presence or absence of glycine (1 mM) for 10 s and transferred to a Wheaton 25 mm vacuum filter system. ³⁶CI^– uptake was terminated by washing twice with ice-cold HEPES buffer through 25 mm glass microfiber filters (Whatman GF/A; Cytiva, Marlborough, MA, USA). Glass fiber filters were placed in scintillation vials, and proteins were solubilized with 1.6 ml of 1 N NaOH for 1 h. EcoLume (8 ml) was added and radioactivity was measured using a liquid scintillation detector (LS-3801; Beckman, Fullerton, CA, USA). In some experiments, neutrophils were pretreated for 30–60 min with strychnine (1 μM) before exposure to 2 μCi/ml ³⁶CI^– in the presence of glycine (1 mM) for 10 s.

The method of separation of viral particles was modified from the original protocol described (107 (link)). Sucrose gradients were prepared fresh before each fractionation with 15% (w/v) and 30 (w/v)% sucrose in 10 mM Tris (pH 7.4)-10 mM NaCl-1.5 mM MgCl2. The Gradient Master gradient maker (Biocomp) was used for continuous 15–30 % gradient preparation. Gradients were prepared in Beckman Ultra-Clear Centrifuge Tubes (14 × 89 mm, Beckman Coulter). Following preparation, 1 mL of radiolabeled lysate was loaded on top of the continuous sucrose gradient and was sedimented via centrifugation for 3h at 27500 rpm at 15°C using a Beckman SW41 rotor in Beckman ultracentrifuge at slow acceleration and deceleration with no brake. After centrifugation, each tube was placed into the Fraction Recovery System (Beckman Coulter) and the bottom of the tube was punctured, 500µL fractions were collected from the bottom of the tube into 1.5mL Eppendorf tubes by gravity flow. Radioactivity in each fraction was calculated by taking 20µL of each fraction and diluting in 4mL of CytoScint Liquid Scintillation Cocktail (MP Biomedicals) with the following measurement in a liquid scintillation counter (Beckman LS-3801).

DNA synthesis was measured by [³H]-thymidine incorporation using 3D ON-TOP assay (Lee et al., 2007 (link)). Briefly, quiesced HRMVECs were trypsinized to a single-cell suspension and 1 × 10⁵ cells in 0.5 ml of the medium were plated onto a matrigel-coated surface of a 24-well plate and VEGFC (100 ng/ml) or vehicle were added to the medium. Cells were allowed to settle for 30 min at 37°C, following which 0.5 ml of medium containing 10% matrigel was added on top of the cells. Six hours later, the cells were pulse-labeled with 1 μCi/ml of [³H]-thymidine for 24 h, at which time cells were washed with cold PBS, collected along with matrigel in 3 ml of 5 mM EDTA in cold PBS into 15 ml centrifuge tubes. The tubes were kept on ice for 15 min to allow the breakdown of matrigel. The cells were then added with 3 ml of 20% (w/v) cold TCA and vortexed vigorously to lyse the cells. The cell mixture was allowed to remain on ice for 30 min and then passed through a GF/F glass microfiber filter. The filter was washed once with 5% cold TCA and once with cold ethanol, dried, placed in a liquid scintillation vial containing the scintillation fluid and the radioactivity was measured in a liquid scintillation counter (Beckman LS 3801). DNA synthesis was expressed as counts/min/well.