Isocitrate dehydrogenase activity assay kit

Cellular lactate levels, α-KG levels, IDH1/IDH2 and IDH3 activities, and NADH/NAD⁺ ratios were measured using the Lactate Assay Kit (Sigma, MAK064), α-Ketoglutarate Assay Kit (Sigma, MAK054), Isocitrate Dehydrogenase Activity Assay Kit (Sigma, MAK062), and NAD/NADH Quantification Kit (Sigma, MAK037), respectively, according to the manufacturer's instructions. The measurements were performed and recorded by a Synergy 2 multidetection microplate reader (BioTek, USA).

Enzymatic activities of the predicted citrate synthase, aconitase and α-ketoglutarate dehydrogenase were tested using the Citrate Synthase Assay kit, Aconitase Activity Assay kit and α-ketoglutarate Dehydrogenase Activity Colorimetric Assay kit, respectively (Sigma-Aldrich, St. Louis, MS, USA). IDH activity assays were performed using the isocitrate dehydrogenase activity assay kit (Sigma-Aldrich, St. Louis, MS, USA) and monitored with a Synergy HT microplate reader (BioTek, Winooski, VT, USA). Reactions were carried out in duplicate at 37 °C in a 96-well plate containing a final volume of 100 µL in each well. Conversion of the isocitrate to α-ketoglutarate was monitored for 30 min following changes in absorbance at 450 nm, corresponding to the production of NADH. A NADH standard curve was constructed, allowing enzyme quantification and calculation of specific activity. In addition, IDH activity was assessed on mature viral particles purified as described above, and on human IDH (Sigma-Aldrich; St. Louis, MS, USA). Initial velocities were calculated using Gen5.1 software (BioTek), and the obtained mean values were fitted using the Michaelis–Menten equation in Prism 6 (GraphPad Software, San Diego, CA, USA).

Citrate synthase and isocitrate dehydrogenase activity were determined with a Citrate Synthase Activity Assay Kit (MAK193) and isocitrate dehydrogenase activity assay kit (MAK062) (Sigma, St. Louis, MO, USA) in accordance with the manufacturer’s instructions.

The Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific, Germany) was used to extract mitochondria of ISKNV-infected or uninfected cells. An Isocitrate Dehydrogenase Activity Assay Kit (Sigma-Aldrich, Germany) and an α-Ketoglutarate Dehydrogenase Activity Colorimetric Assay Kit (Sigma-Aldrich, Germany) were used to detect the enzyme activity in cells. Briefly, mitochondria from 5 × 10⁶ cells were homogenized in 300 μL of assay buffer to prepare sample solution. Twenty μL of sample solution was pipetted into the wells of a 96-well plate. Then, a reaction mixture (8 μL developer, and 2 μL α-KGDH substrate or 2 μL IDH substrate) was added to each well. For IDH2 activity analysis, 2 μL of NADP+ was added additionally to the reaction mixture. After 3 min, the absorbance at 450 nm was measured every minute in a microplate reader (Infinite M200 Pro, Tecan, Switzerland) until the value of the most active sample is greater than the value of the highest standard. IDH2 activity was defined as the production of 1.0 μM NADPH per minute. α-KGDH activity was defined as the production of 1.0 μM NADH per minute. Three replicates of each sample were performed. For samples exhibiting significant background, we included a sample blank by omitting the substrate.