Microtiter plate

Phenoloxidase (PO) activity was monitored spectrophotometrically as the formation of dopachrome from 3,4-dihydroxyDL-phenylalanine (DL-DOPA, Sigma-Aldrich) as reported previously by Giglio et al. (2018) (link), with minor modifications. Briefly, 20 μL of plasma were mixed with either 180 μL of DL-DOPA (3 mg/mL in PBS) or 180 μL of a solution of DL-DOPA (3 mg/mL in PBS) and SDS (1 mg/mL) in a microtiter plate (Biorad), for the determination of basal and total plasmatic PO (proPO) enzyme activity, respectively. SDS has been described as a good chemical activator of PO from its inactive zymogen, prophenoloxidase (proPO, Radha et al., 2013) (link). Absorbance was measured kinetically at 25 °C at 492 nm over 60 min at 5-min intervals in an Infinite® 200 PRO microplate reader (Tecan, Männedorf, Switzerland) equipped with the software Tecan i-control (version 1.7.1.12). Four technical repetitions were performed for each plasma sample. The enzyme activity was measured as the slope (absorbance vs time) of the reaction curve during the linear phase of the reaction. Absorbance values were blank (reagents) subtracted. The slope of the reaction curve at V max was plotted as absorbance per μL of hemolymph per min.