Citrate synthase assay kit

Citrate synthase activity (U•μg⁻¹•min⁻¹) in the gastrocnemius muscle (n = 10/group) was measured from the same muscle homogenates that were used to determine the total protein content (Citrate Synthase Assay Kit Sigma-Aldrich) with an automated KoneLab instrument (Thermo Scientific).

Five whole flies were homogenised for 60 s in 100 μl CellLytic MT Cell Lysis Reagent (Sigma) and debris was cleared from the lysate by centrifugation at 10,000x g for 10 mins at 4 ^oC. The lysate was diluted 5-fold in lysis reagent and used for both citrate synthase and BCA protein content assays.
Citrate synthase (CS) activity was assayed using the Citrate Synthase Assay Kit (Sigma) on a 96 well plate according to manufacturer instructions, with each sample measured in triplicate. CS is an enzyme which catalyses a key reaction in the TCA cycle, where citrate is produced from acetyl CoA and oxaloacetate. A by-product of this reaction is coenzyme A (CoA-SH), which reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) in this assay to produce 2-nitro-5-benzoic acid (TNB). The concentration of TNB in each sample was measured by light absorbance at 412 nm. A FLUOstar plate reader (Omega) was used to monitor the change in absorption of 412 nm wavelength light after addition of oxaloacetate to each sample and the rate of change in absorbance during the linear phase of the reaction was used to calculate CS activity (μM / ml / min). CS activity of each sample was normalised to total protein content, quantified by BCA assay (Thermo Scientific, UK).

A commercially available kit (Citrate Synthase Assay Kit, CS 0720, Sigma), was used according to manufacturer's instructions, to determine citrate synthase (CS) activity in the frozen samples.

Cells were treated for 24 h with 10 µM NCT-503 or inactive NCT-503 control, and then lysed in CelLytic M provided in the citrate synthase assay kit (CS0720, Sigma-Aldrich, St. Louis, MO, USA). Aliquots of whole-cell extract containing equal total protein amounts were used for the citrate synthase activity assay according to the manufacturer’s instructions. Total protein was assessed by colorimetric protein quantification based on the Bradford assay, ROTI®Quant (Carl Roth, Karlsruhe, Germany).

Mitochondria isolated using the MACS Mitochondria Isolation Kit (Miltenyi) were examined for intactness using a citrate synthase assay kit (Sigma-Aldrich) according to manufacturer’s instructions. Briefly, isolated mitochondria from approximately 10⁷ HeLa cells were incubated at 37°C for 0 minutes or 6 hours, then were separated into two equal aliquots, one in which mitochondria were left intact, and one in which mitochondria were lysed. Because citrate synthase activity is restricted to the mitochondrial matrix, inner mitochondrial membrane intactness can be assessed by comparing the citrate synthase activity in a sample of intact mitochondria to that of a sample of lysed mitochondria using an indicator dye detected spectrophotometrically. This mitochondrial intactness assay indicated that, after 6 hours of incubation in mitochondrial secretion assay buffer, the percent of mitochondria that were ruptured rose from 19.0% at 0 minutes to 29.5% at 6 hours (data not shown). These data indicate that mitochondria isolated in our laboratory are largely intact, even after several hours.