Aconitase activity assay kit

Cells were transfected and treated with indicated drugs for 24 hours and then collected. Hydroxyl radicals were detected using a Hydroxyl Radical Assay Kit (Jiancheng, Nanjing, China), ATP was measured using an ATP assay kit (Beyotime Biotechnology, Shanghai, China), cell oxygen consumption rate (OCR) was measured using Mito‐Xpress and pH‐Xtra kits (Luxcel Bioscience, Cork, Ireland), mitochondria extraction from cultured cells was performed (MP‐007, Inventbiotech), ACO2 activity was determined using an Aconitase Activity Assay Kit (MAK051, Sigma), and isocitrate and citrate concentrations were determined using an IsoCitrate Assay Kit (MAK319, Sigma) and a Citrate Assay Kit (MAK057, Sigma), respectively. All assays were performed according to the manufacturers' protocols.

Mitochondrial aconitase 2 activity was measured by Aconitase Activity Assay Kit (Sigma-Aldrich, MAK051) according to the manufacturer’s protocol.

Protein extracts were obtained from groups of twenty DJ-1β mutant and control female flies, as described in [7 (link)]. Aconitase (Aco; EC 4.2.1.3) activity was measured with the Aconitase Activity Assay Kit (#MAK051; Sigma-Aldrich, St. Louis, MO, USA), following manufacturer’s instructions, and assays were performed in triplicate. Succinate dehydrogenase (SDH; EC 1.3.5.1) activity was measured using a protocol adapted from [39 (link)]. First, mitochondrial extracts were obtained from groups of 30 DJ-1β mutant and control female flies, as described in [40 (link)]. Then, 50 µL of mitochondrial extracts were added to 50 µL of assay buffer (4 mM sodium azide, 50 µM 2,6-dichlorophenolindophenol and 2 µg/mL rotenone) and transferred to a 96-well plate. The reaction was started by adding succinate to a final concentration of 10 mM. Absorbance was measured at 600 nm using an Infinite 200 PRO reader (Tecan) every 30 s for 20 min at 25 °C. Sample absorbance levels were measured, subtracting their corresponding blanks. Assays were performed in triplicate.

The enzyme activities (U) of both aconitase and SDH within PB MNC homogenates were determined using the Aconitase Activity Assay Kit and Succinate Dehydrogenase Activity Colorimetric Assay Kit (MAK051 and MAK197; both Sigma-Aldrich, UK) according to the manufacturer’s instruction. Absorbance was measured using a FLUOstar OPTIMA plate reader (BMG Labtech) and OPTIMA software (BMG Labtech; version 2.20R2). Homogenate total protein concentration was quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). Enzyme activity values were calculated as activity per 1 µg of total protein.

Relative mitochondrial aconitase activity was quantified using the colorimetric Aconitase Activity Assay Kit from Sigma (#MAK051), following the manufacturer’s instructions as previously described (Martelli et al., 2020 (link)). A total of six biological replicates (25 whole larvae per replicate) were exposed to 2.5 ppm spinosad for 2 hr, whilst six control replicates (25 whole larvae per replicate) were exposed to DMSO for 2 hr. Absorbance was measured at 450 nm in a FLUOstar OPTIMA (BMG LABTECH) microplate reader using the software OPTIMA and normalized to sample weight. The data were analyzed using one-way ANOVA followed by Tukey’s honestly significant difference test.