Colorimetric fluorometric assay kit

It is the indicator of lipid peroxidation (Colorimetric/Fluorometric Assay Kit, Catalog # K739-100, BioVision, USA). 10 mg of the sample tissue was blended with 150 μl dH2O, 3 μl BHT, and 1 vol of 2 N perchloric acid, vortexing then centrifuged to remove precipitated protein for 10 min (4000 rpm).

Triglyceride (TG) content was determined using a colorimetric/fluorometric assay kit (Biovision, Milpitas, CA, USA). 3T3-L1 cells were seeded into a 96-well plate and differentiated with CTE (500–1000 µg/mL) until they became mature adipocytes. A lipid droplet was then extracted by extraction buffer and converted by lipase to glycerol and fatty acid. A wavelength of 570 nm was used to measure the released glycerol.

ROS and LPO assays were performed as described previously with some modifications [27 (link)]. The principle of ROS assay is the formation of 2′7′ dichlorofluorescein (DCF) from the oxidation of 2′7′-dichlorodihydrofluorescein. In ice-cold buffer, the cortex and hippocampus homogenates were diluted to yield 2.5 mg tissue/500 μL as a final concentration. Then, 1 mL of Locke’s buffer mixture (pH ± 7.4), 0.2 mL of homogenates, and 10 mL of 5 mM 7-dichlorodihydrofluorescein diacetate (DCFH-DA) were incubated for 15 min at room temperature to form fluorescent DCF from DCFH-DA. The formation of DCF from DCFH-DA was evaluated via a microplate reader at 484 nm (excitation) and 530 nm (emission). In the absence of homogenate, to enable calculation of DCF formation (background fluorescence), we measured parallel blanks. ROS levels are expressed as DCF formed (pmol)/amount of protein (mg). For further determination of oxidative stress, the level of LPO was quantified. Cortex and hippocampus of adult mice were homogenized and free malondialdehyde (MDA), a specific marker for LPO, was measured with a colorimetric/fluorometric assay kit according to the manufacturer’s instructions (BioVision, San Francesco, CA, USA, Cat#739-100).

ATP levels were measured with a colorimetric/fluorometric assay kit (BioVision). Samples were processed and analyzed according to the manufacturer’s protocol. Standard curve was generated and sample absorbance was measured accordingly.

Mouse studies were approved by the Italian Ministry of Health. Mice were group-housed (2–5 mice per cage) in standard mouse cages and maintained under a 12 h light/dark cycle with free access to water and standard mouse chow. Wild-type six-week-old male C57BL/6N mice (weight: 20–22 g) purchased from Charles River Laboratories were injected intraperitoneally (i.p.) with 0.5 μg/g of body weight of CD95-activating antibody (CD95-Ab; BD Biosciences), 0.6 μg/g of body weight of α-amanitin (Sigma-Aldrich), 0.4 mg/g of body weight of acetaminophen (APAP) (Sigma-Aldrich), or vehicle (saline). 20 mg/kg/day of garcinol (Enzo Life Sciences) or vehicle (saline) was injected i.p. for five days prior to the injection of CD95-Ab. Galloflavin at the doses of 10, 20, 40 or 80 mg/kg diluted in 40% dimethyl sulfoxide (DMSO) or vehicle (40% DMSO) was injected i.p. at the same time as CD95-Ab, α-amanitin, or APAP injections and was continued daily until mouse sacrifice by cervical dislocation at 72 h after CD95-Ab, α-amanitin, or APAP injections. Blood samples were collected by retro-orbital bleedings and were analyzed for alanine aminotransferase levels by colorimetric/fluorometric assay kit (Biovision) according to manufacturer’s instructions.