Succinate dehydrogenase activity colorimetric assay kit

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.

SDH activity was measured using the Succinate Dehydrogenase Activity Colorimetric Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions. For each sample, 4 frozen whole male flies or 25 isolated heads were rapidly homogenized in a 1.7 ml Eppendorf tube with 100 µl of ice-cold SDH assay buffer using a motorized pestle homogenizer (Kontes), kept on ice for 10 min, and centrifuged at 10,000 g for 5 min at 4 °C. The supernatant was transferred to a fresh tube. In a clear 96-well plate, 10 µl of supernatant was mixed with 92 µl of reaction mix (88 µl of SDH assay buffer, 2 µl of SDH substrate, and 2 µl of SDH probe) in each well. The absorbance was immediately read at 600 nm in kinetic mode for 30 min at 25 °C using a BioTek Synergy 2 microplate reader. The SDH activity was calculated according to the manufacturer’s instructions and normalized to total protein concentration of the sample.

Subcellular fractions were analyzed for the presence of markers beta-galactosidase (cytoplasm), succinate dehydrogenase (inner membrane), and 3-deoxy-D-manno-oct-2-ulosonic acid or KDO (outer membrane) (S1 Fig). Fractions were adjusted to 10 μg protein content. Beta-galactosidase activity was measured using the Beta-galactosidase Enzyme Assay System (Promega) according to the manufacturer’s instructions. Succinate dehydrogenase activity was measured using the Succinate Dehydrogenase Activity Colorimetric Assay Kit (Sigma-Aldrich) per the manufacturer’s instructions, except samples were incubated on ice in SDH Assay buffer for 6 h prior to starting the assay. The concentration of KDO was determined as described previously [28 (link)].

Mitochondria were isolated according to the protocol previously described [30 (link)]. The SDH activity was determined using the Succinate Dehydrogenase Activity Colorimetric Assay Kit (Sigma–Aldrich Inc., St. Louis, MO, USA). It was measured by monitoring the decrease in absorbance at 600 nm for 10 min in the presence of 0.1 M sodium succinate (pH 7.5), 0.05 mM 2,6-dichlorophenolindophenol, 0.1 M NaPO₄ buffer (pH 7), and 50–100 μg of resuspended biological membranes in a 1 mL reaction volume. SDH activity was expressed relative to the background reduction of 2,6-dichlorophenolindophenol (membranes without succinate).