Gpdh activity assay kit

The 3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM), glutamine, penicillin-streptomycin, fetal bovine serum (FBS), and TRIzol reagent were obtained from Invitrogen (Carlsbad, CA, USA). The cell count kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). An assay kit for TG was obtained from Asan Pharmaceutical Co. (Seoul, Korea). The GPDH activity assay kit was from Takara (Kyoto, Japan). The bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific (Pittsburgh, PA, USA). Universal SYBR^® Green PCR Master Mix was purchased from Qiagen (Chatsworth, CA, USA). M-MLV reverse transcriptase was purchased from Promega (Madison, WI, USA). The NO assay kit was purchased from Thermo Scientific (Pittsburgh, PA, USA).

GPDH activity was measured using a GPDH activity assay kit (Takara, Kyoto, Japan) in accordance with the manufacturer’s instructions [19 (link)]. The 3T3-L1 adipocytes were incubated for 7 days with 0, 0.1, 0.5, or 1 μg/mL of HCF. GPDH activity was measured by monitoring the absorbance at 340 nm using a microplate reader and normalized to the cellular protein content.

For lipid accumulation, Oil Red O staining was performed for 3T3-L1 cell lines. Mature adipocytes were incubated in complete media, with or without 5-uRCK or ellagic acid, for 72 h, and the cells were fixed with 4% formaldehyde for 20 min. Then, cells were washed with 60% isopropanol and fully dried. Oil Red O working solution was added, and cells were maintained at 30 min at room temperature. Mature fat cells were stained with Oil Red O solution. Excess stain was removed by washing thoroughly with distilled water. Fully differentiated mature fat cells were photographed using an inverted microscope (Nikon Instrument, Melville, NJ, USA). Differentiation was monitored using microscopy and quantified via elution with 100% isopropanol. Optical density (OD) measurements were obtained at 490 nm. GPDH activity was measured with a GPDH activity assay kit (Takara Bio Inc., Shiga, Japan) in accordance with the manufacturer’s instructions. Total triglyceride content in the mature adipocytes was measured; 100% isopropanol was added to mature adipocytes and incubated at room temperature for 10 min followed by measurement of absorbance at 630 nm using a TG assay kit (Asan pharmaceuticals, Seoul, Korea). Values for TG levels were expressed as a percentage of the control.

To measure triglyceride levels and GPDH activity, 3T3-L1 preadipocytes (5 × 10⁴ cells/well) were seeded in 24-well cell culture plates and incubated in 10% CS-DMEM. After reaching confluence, the cells were cultured with insulin (1 μg/mL) in the presence of baccharin in 10% FBS-DMEM for 9 days. The cells were washed twice with ice-cold PBS, harvested in ice-cold 25 mM Tris-HCl (pH 7.5) buffer containing 1 mM ethylenediaminetetraacetic acid, and lysed by brief sonication. The triglyceride levels of the lysate were quantified using a Triglyceride E-test kit (Wako) and expressed as mg/mg protein. GPDH activity was measured using a GPDH Activity Assay Kit (Takara Bio, Shiga, Japan) and expressed as mU/mg protein. Protein concentration was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Tokyo, Japan).

Cells cultured in 12-well plates were washed with PBS and harvested at 1, 3 and 6 days of induction. GPDH activity was measured using the GPDH Activity Assay Kit (Takara) according to the manufacturer's instructions. Protein concentrations of cell culture homogenates were determined by the method of Lowry et al. [9 (link)] using BSA as standard.