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Hep G2 cell line was purchased from American Type Culture Collection. Dulbeccos Modified Eagle Medium, 0.5 Trypsin-EDTA 10x, and Penicillin-Streptomycin (PS) were obtained from Invitrogen Corporation (NY, USA). Fetal Bovine Serum (FBS) was gotten from Welgene Inc. (Daegu, South Korea). Fatty acids (Palmitic, Oleic and Dedocanoic acid), Dimethyl sulfoxide (DMSO) and Tween 20 came from Sigma (MO, USA). Bovine serum albumin (BSA) was from Santa Cruz Biotechnology (CA, USA). MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) was purchased from Molecular Probes (Oregon, USA). LDH assay (Lactate dehydrogenase assay) was from ROCHE (Mannhein, Germany). [email protected]/503 and Carboxyl-H2DCFDA were purchased from Invitrogen Corporation (Oregon, USA). Nile red was from Fluka (MO, USA). Triglyceride Quantification Kit and ATP Colorimetric/Flourometric Assay Kit were purchased from BioVion Inc. (CA, USA). Annexin V Floustaining kit was from Roche (IN, USA). Phosphate buffered saline was made up of chemicals at pH 7.4, including 11.7g NaCl, 5.5g Na2HPO4-7H2O, and 1.35g NaH2PO4. All other chemicals met in standard grade of analysis.
Culture of HepG2 cells
HepG2 cells were cultured in Dulbecco's modified Eagle's medium, containing 10 (v/v) fetal bovine serum and 1 (v/v) Penicillin-Streptomycin under 5 CO2, 95 humidity at 37°C. The cells were subcultured by using 0.5 Trypsin-EDTA 1x (Invitrogen Corporation, NY, USA) for detachment and seeded at 1x105 cell/ml in all experiment.
Fatty acid treatment
When 80 confluency of HepG2 was reached, it was treated with various concentrations of the fatty acids (0 mM, 0.25 mM, 0.5 mM, 1.0 mM and 2.0 mM) for 24 h. The stock solution of fatty acids was prepared at 100 mM by dissolving in DMSO and stored at -200C. The stocks were diluted in DMEM media containing 1.0 bovine serum albumin (BSA) to obtain working solution in all experiments.
Cytotoxicity was based on the measurement of cytoplasmic enzyme activity by using cytotoxicity detection kit (ROCHE, Mannhein, Germany). The cytoplasmic enzyme was released from damaged cells that its enzyme activity expresses to the proportion of toxiced-cell. Lactate dehydrogenase (LDH) presents in all cells which is a stable cytoplasmic enzyme. When the membrane integrity of the cells is damaged, it is quickly released into the media. In this assay, NAD+ is reduced to NADH/H+ during conversion of lactate to pyruvate by the LDH-catalyzed. After that, H/H+ from NADH/H+ was transferred by the catalyst (diaphorase) to the tetrazolium salt (yellow) which was reduced to formazan (red). To conduct the assay, the culture supernatant is collected cell-free after desire exposure time (24 h). The reaction mixture from the kit was then applied in the samples. The absorption of the formazan dye formed was measured at 490 nm on an ELISA reader (VERSARMAX, Molecular Divices., CA, USA).
Cell viability was measured based on measurement absorption of a water-insoluble purple formazan which was reduced from a yellow water-soluble tetrazolium salt in live cells. Briefly, the cells were treated with MTT (5 mg/ml) in DMEM at 37 0C for 1.5 h. Then, the media were removed, and DMSO was added to dissolve the furmazan crystals. After gently pipetting, the absorbency was measured at 570 nm using an ELISA reader (VERSARMAX, Molecular Divices., CA, USA). The estimation of cell viability was calculated by comparing between the spectra value of treated and untreated cells.
Quantification of triglyceride
Triglyceride content (TG) was determined according to an enzymic method (BioVion Inc, CA, USA). In this method, glycerol is a product by TG-catalyzed which reacts with the probe to generate coloration measured on spectrophotometry at 570 nm. In briefly, the cells were washed twice times with cold PBS, then homogenized in 5 Triton-X100 solution. After slowly heating at 80-100°C for 5 min, the samples were centrifuged at 12000 rpm for 5 min. The supernatant collected from removing insoluble materials was added 2 μl of lipase, mixed well and incubated for 20 min at room temperature. Finally, 50 μl of the reaction mix was putted in each sample for 45 min of incubation, protected from light. The value of triglyceride content was quantified based on triglyceride standard curve that was constructed with different concentrations of TG (0, 20, 40, 60, 80, and 100 nmol/ml).
Measurement of reactive oxygen species (ROS) generation
The measurement of ROS production within cells was carried out by using 2′,7′-Dichlorohydrofluorescein diacetate (Carboxyl-H2DCFDA; Invitrogen Corporation, Oregon, USA) which is combined into fluorescent products in the presence of H2O2 and other ROS molecules and esterases (Zhenyuan Song et al, 2007). After the cells were overloaded with 1.0 mM fatty acids, 10 mM final concentration of Carboxyl-H2DCFDA was added in the media without FBS at 370C in darkness for 30 min. Then, the cells were washed twice times with warmed PBS and lysed in 200ïl RIPA buffer (PIERCE, IL, USA). The lysed-cells were centrifuged at 12000 rpm for 5 min. The supernatants were conveyed to a 96-well back plate which were excited at 485 nm and emitted at 530 nm for the Carboxyl-H2DCFDA fluorescence on Fluorometer (VICTOR2, Perkin Elmer., MA, USA).
ATP quantification assay
ATP Colorimetric/Fluorometric Assay Kit (BioVion Inc, CA, USA) was used to measured ATP content. The principle of this kit is to quantify a product that was generated by the phosphorylation of glycerol by colorimetric methods. As the procedure of the assay, the cells were lysed in 200 μl of ATP Assay Buffer. The lysed-cells were centrifuged at 15000 g for 2 min at 40C. The supernatant was collected and transferred to a 96-well plate. The Reaction mix from the kit was added to each well, and then the samples were incubated for 30 min at room temperature, protected from light. When completion of the incubation time, the absorption of the samples were determined at wavelength of 570 nm on an ELISA reader (VERSARMAX, Molecular Divices., CA, USA).
Detection of late apoptosis and trilyceride accumulation by Confocal
HepG2 seeded in the 24-well plate and treated with final concentration of fatty acids to 1.0 mM for 24 h. After the incubation time, the cells were washed twice times with PBS. Then, Bodipy @493/503 (Invitrogen, Oregon, USA) was dissolved in PBS at 5 ïg/ml which was added into each well. This process was kept in darkness for 15 min at 370C. After that, the Bodipy solution was removed and the cells were then washed by Binding buffer from Annexin V Floustaining kit (Roche, IN, USA). Finally, the cells were incubated in 100 ïl/ml of Propidium iodide (PI) for 10 min in darkness. Exposition of TG accumulation and apoptosis was observed at excitation of 488 and 543 nm, and emission of BP 505-530 and LP 650 nm on Carl Zeiss Confocal Microscopy (LSM 510 meta, Carl Zeiss., Jena, Germany), respectively.
ROS and trilyceride staining on Confocal
ROS generation in HepG2 was stained by using Carboxyl-H2DCFDA, whereas Nile red (Fluka., MO, USA) was used to capture TG fluorescence on Confocal microscopy. In this experiment, the cells were prepared as above. Before the dyes treatment, the cells were washed with warmed PBS twice times. The carboxyl-H2DCFDA was applied at 10mM final concentration in Serum free media (DMEM without FBS), and incubated for 30 min at 370C, protected from the light. Nile red was then added at 10 mM, and 10 min of incubation at 370C after the cells were rinsed with warmed PBS again. Zeiss LSM Image Brown software (LSM 510 meta, Carl Zeiss., Jena, Germany) was handled to take ROS and TG image at excitation of 488 and 543 nm and emission of BP 530-600 nm and BP 585-615 nm, respectively.
All results were expressed as mean of repeated three or four value ± SEM. The difference between groups was identified by using t.test. p < 0.05 was considered statistical significant.