Varioskan lux 96 well plate reader

Enzyme activity of general esterases was assessed using p-nitrophenylacetate as substrate, following a previous procedure [43 (link)]. In brief, the protein homogenate (10 µL) was mixed in a microtiter plate well with 1 mM p-nitrophenyl acetate (200 µL) in 50 mM phosphate buffer pH 7.4. The absorbance was measured at 405 nm after a 10 min incubation period using a Varioskan LUX 96-well plate reader (Thermo Fisher Scientific, Waltham, MA, USA). The specific esterase activity was calculated using the kinetic slope to convert μmoles of product per minute (6.53 mM⁻¹cm⁻¹ and 0.3 cm), thereby determining the specific enzyme activity expressed as μmol of substrate/min/mg protein.

The Ellman method was employed to assess AChE activity in both untreated and Z. schreberi-treated mites, following a previous procedure [41 (link)]. In brief, protein homogenate (20 µL) was mixed in a microtiter plate well with 1% 100-X triton phosphate buffer (pH 7.8, 145 µL), acetylthiocholine iodide (0.01 M, 25 µL), and 5,5′-dithiobis-(2-nitrobenzoic acid) (0.01 M, 10 µL). This procedure, conducted with three biological and three technical replicates, monitored the enzyme reaction kinetics at 415 nm after a 10 min incubation period using a Varioskan LUX 96-well plate reader (Thermo Fisher Scientific, Waltham, MA, USA). By applying an extinction coefficient of 13.6 mM⁻¹cm⁻¹ and a path length of 0.3 cm, the kinetic slope was converted to μmoles of product per minute, thereby determining the specific enzyme activity expressed as μmol of substrate/min/mg protein.

GST activity was assessed using 1-chloro-2, 4-dinitrobenzene (CDNB) as the substrate, following a previous procedure [42 (link)]. Briefly, the reaction was initiated by mixing CDNB (50 mM in ethanol, 50 µL) and reduced glutathione (50 mM in 0.1 M phosphate-buffered saline (PBS), pH 6.5, 150 µL) in a 5 mL vial with phosphate buffer (0.1 M, pH 6.5 containing 1 mM EDTA, 2.78 mL). Subsequently, 20 µL of protein homogenate was added, and the solution was thoroughly mixed. An aliquot (200 µL) from the resulting mixture was dispensed into a microtiter plate well for measurement, with the procedure performed in triplicate for both biological and technical replicates. The absorbance of each reaction mixture at 340 nm was then recorded using kinetics on a Varioskan LUX 96-well plate reader (Thermo Fisher Scientific, Waltham, MA, USA) after a 10 min duration. The specific GST activity was calculated using the kinetic slope to convert μmoles of product per minute (9.6 mM⁻¹cm⁻¹ and 0.3 cm), thereby determining the specific enzyme activity expressed as μmol of substrate/min/mg protein.