Daf 2

Cancer cells in suspension were washed with PBS and loaded with 50 μM H2DCFDA (Molecular Probes, Life Technologies, D399) or 5 μM DAF-2 (Cayman Chemical, Catalog # 85165) for 30 min at room temperature in the dark. Cells were washed twice with PBS and treated with various doses of Ivermectin in R10 medium. Cells were incubated for 1 h in 5 ml FACS tubes at 37 °C and 5% CO₂, and immediately analyzed by flow cytometry. Generation of intracellular ROS and NO results in corresponding increases in the GFP fluorescence of the H2DCFDA and DAF-2 probes, respectively.

Reactive nitrogen species produced in tick tissues were detected with the fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2) purchased from Cayman Chemical (Ann Arbor, MI, USA). DAF-2 reacts with NO in the presence of oxygen to produce triazolofluorescein (DAF-2T). Briefly, salivary glands and midguts were dissected from unfed adult I. scapularis female ticks, or from adult female ticks fed to repletion on New Zealand White rabbits. Salivary glands and midguts were rinsed extensively in PBS, and transferred to individual 1.5 ml microcentrifuge tubes with 25 μM DAF-2 for 10 min at RT. Following incubation, tick tissues were placed in individual pools of ~10 μL PBS on glass microscope slides, and imaged using a Nikon Eclipse E800 epifluorescence microscope (Nikon USA, Melville, NY, USA) by digital interference contrast (DIC) microscopy or fluorescence microscopy using a FITC filter (Ex_λ = 495 nm, Em_λ = 519 nm).

ECs were seeded at 5 × 10⁵ cells/well in 6-well plates and cultured for 2 days until confluent in complete EBM-2. Sixteen hours before the assay, ECs were serum-depleted in EBM-2 containing 0.2% FBS. On the day of the assay, ECs were incubated with 100 μg/mL of HDL in serum-depleted EBM-2 medium containing 1 μM of 4,5-diaminofluorescein diacetate (DAF2, Caymanchem) at 37 °C. After 6 h, ECs were washed with PBS, trypsinized, and triazolofluorescein fluorescence was measured (excitation wavelength of 485 nm, emission 538 nm), using an Infinite M200Pro plate reader (Tecan). In addition to DAF2 measurement, the phosphorylation of eNOS at Ser1177 was compared to total eNOS by immunoblotting (below) in cell lysates harvested 15 min after HDL treatment.