Spectramax paradigm reader

Twenty adult planthoppers were homogenized in 100 μl of 10 mM Tris-HCl buffer (pH 8.0) and centrifuged at 12,000 × g for 15 min at 4°C, and 65 μl of the supernatant was gently mixed with 100 μl of 4 mg/ml dopamine in 10 mM Tris-HCl buffer (pH 8.0) in a 96-well plate at 27°C for 10 min. Twelve replicates for viruliferous and nonviruliferous planthoppers were prepared. For analysis of PO activity from hemolymph, 65 μl of the clear hemolymph solution was mixed with 100 μl of 4 mg/ml dopamine. The absorbance of melanin was monitored at 490 nm (A₄₉₀) by the use of a SpectraMax Paradigm reader (Molecular Devices, San Jose, CA, USA) every 5 min. The protein concentration of the supernatant was determined using the Bradford method. One unit (U) of PO activity was defined as 0.001 ΔA₄₉₀ for every milligram protein in 1 min (39 (link)). Values representing the PO activity of each group are represented as means ± standard errors (SE). Differences were statistically evaluated in SPSS 17.0 using Student’s t test to compare two means or using one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons.

Human Brain Microvascular Endothelial Cells (hBMECs) were seeded on plate wells at a concentration of 3 × 10⁵ cells/mL. The monocytes were obtained from individuals other than those who composed the monocyte pool of the proteomic analysis were used in the adhesion and transmigration assays. Monocytes were labelled with 1 µM CFSE (Invitrogen) for 15 minutes, washed three times and resuspended in medium supplemented with 10% FCS. 2 × 10⁵ cells were transferred onto hBMECs monolayers and incubated for 3 h at 37 °C under 5% CO₂ in humidified atmosphere. Adherent cells were quantified by lysing cells with 0.1 M NaOH and measuring the fluorescence intensity using SpectraMax Paradigm reader (Molecular Devices). Data are expressed as mean ± SEM of arbitrary units.
Monocyte transmigration assays were performed using Millipore Millicell inserts (12 mm diameter, 8 µm pore size). hBMEC were seeded on the upper side of the filter at a concentration of 3 × 10⁵ cells/insert, and monolayers reached confluence. 2 × 10⁵ CFSE-labelled monocytes were transferred onto monolayers and incubated for 12 h at 37 °C under 5% CO₂ in humidified atmosphere. Transmigrated monocytes were recovered and counted by flow cytometer using 40,000 Flow-Check Fluorospheres (Beckman Coulter).