The concentration of acetyl-CoA was detected by using an Acetyl-CoA assay kit (Sigma MAK039)
according to manufacturer's protocols. Brie y, cells or tissues were washed with cold PBS, harvested in RIPA buffer (Beyotime Biotech Co. Ltd., China), and then sonicated. The cellular or tissue protein was precipitated with PCA (BioVision K808-200). After centrifugation at 13000 g for 2 minutes, supernatant was recovered and neutralized by the addition of potassium bicarbonate until the pH of the sample was in the range of 6-8 and precipitates were removed by centrifugation. The supernatant was used to measure acetyl-CoA concentration in triplicate by using uorescence assay method. Fluorescence intensity was measured (λ ex = 535/ λ em = 587 nm) in black 96-well at-bottom plates with clear bottoms.
The concentration of citrate was measured by using a citrate colorimetric assay kit (BioVision K655-100) according to manufacturer's protocols. Brie y, cells were rapidly homogenized with citrate assay buffer, then centrifuged at 15000 g for 10 minutes to remove cell debris. The cellular protein was precipitated with PCA (BioVision K808-200). The sample was added into duplicate wells of a 96-well plate to bring the volume to 50 μL with a citrate assay buffer. After incubation at room temperature for 30 minutes, optical density (OD) value was measured at 570 nm.
according to manufacturer's protocols. Brie y, cells or tissues were washed with cold PBS, harvested in RIPA buffer (Beyotime Biotech Co. Ltd., China), and then sonicated. The cellular or tissue protein was precipitated with PCA (BioVision K808-200). After centrifugation at 13000 g for 2 minutes, supernatant was recovered and neutralized by the addition of potassium bicarbonate until the pH of the sample was in the range of 6-8 and precipitates were removed by centrifugation. The supernatant was used to measure acetyl-CoA concentration in triplicate by using uorescence assay method. Fluorescence intensity was measured (λ ex = 535/ λ em = 587 nm) in black 96-well at-bottom plates with clear bottoms.
The concentration of citrate was measured by using a citrate colorimetric assay kit (BioVision K655-100) according to manufacturer's protocols. Brie y, cells were rapidly homogenized with citrate assay buffer, then centrifuged at 15000 g for 10 minutes to remove cell debris. The cellular protein was precipitated with PCA (BioVision K808-200). The sample was added into duplicate wells of a 96-well plate to bring the volume to 50 μL with a citrate assay buffer. After incubation at room temperature for 30 minutes, optical density (OD) value was measured at 570 nm.