Bc0590

The free fatty acid detection test was carried out with reference to the kit instructions (Solarbio Life Sciences, BC0590, China). The well-grown cells were collected by removal of used medium, washed with PBS once, planted in a six-well plate, by supplementing 200 μL of western and IP cell lysis buffer to each well, lysed for 5 s, and collected using an appropriate centrifuge tube after lysis. The cells were centrifuged at 10,000g for 10 min to collect the supernatant for the determination.

Removed 50 μL of hemolymph from larvae and pupae at various developmental stages and mixed with phenylthiourea (PTU) to prevent hemolymph melanization, added reagents as directed (BC0590, Solarbio, Shanghai, China), and measured absorbance at a wavelength of 550 nm by spectrophotometry. The standard curve was created by diluting the standard to various concentrations, and the FFA levels were estimated using the standard curve.

TLC analysis was performed in accordance with a previous method with some modifications [57 (link)]. The total lipid extracts dissolved in chloroform were separated through TLC (silica gel GF254, 25 mm × 75 mm, 0.25 mm thickness, Qingdao Haiyang, China) in a mixture of hexane/diethyl ether/acetic acid (80:20:2, v/v/v) until the layers separated. The plate was stained with iodine vapor at 37 °C for 10 min to separate TAG, DAG, FFAs, and PLs.
FFA content was determined by using a FFA content detection kit (BC0595, Solarbio, China). Data were analyzed in accordance with the instruction manual. The determination principle is that under weakly acidic conditions, FFAs react with copper salt to produce fatty acid copper salt, and a characteristic absorption peak appears at 550 nm. Therefore, content of FFAs has a linear relationship with the degree of color development. TAG content was determined with a TAG content detection kit (BC0590, Solarbio, China) in accordance with a previous study [58 (link)].