Free fatty acid quantification colorimetric fluorometric kit

Total fatty acid quantification was performed with a Free Fatty Acid Quantification Colorimetric/Fluorometric Kit (Biovision, Mountain View, CA, USA) according to the manufacturer’s instructions. Brain tissues were separated into hippocampus and cortex, and lysed. The protein concentration was measured using a BCA method. Samples were incubated with acetyl-CoA synthetase reagent, enzyme mix, and enhancer. Mixtures were incubated for 30 min and Total FA levels were detected using a microplate reader (at 550 nm).

Free fatty acid quantification was performed using a free fatty acid quantification colorimetric/fluorometric kit (BioVision).26 The palmitic acid standard liquids were diluted to 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well. 1 × 10⁶ SACC‐LM or SACC‐83 cells or 10 mg SACC tissue samples were extracted by homogenization with 200 µL of chloroform‐Triton X‐100. The extracts were spined 10 minutes and dried to remove trace chloroform. The dried lipids were dissolved in 200 µL of fatty acid assay buffer. The enough reagents were mixed for the number of assays and standard performed according to the manufacturer's instructions. OD 570 nm was used for colorimetric assay or Ex/Em = 535/590 nm was used for fluorescence in a microplate reader.
In SACC tissues, the FFA concentration of the normal salivary gland tissues was designated as the relative baseline. Compared with the baseline, the FFAs concentration was divided into the low FFAs group and the high FFAs group.

Neonatal ventricles were homogenized with 1% Triton X-100 in chloroform. The total lipids were at the organic phase, separated by centrifugation at 12,000 × g for 15 min. Total lipids were air-dried at 50 °C until pellets formed and then were subjected to vacuum to remove trace chloroform. The amount of free fatty acid was determined using a free fatty acid quantification colorimetric/fluorometric kit (BioVision).