Hep G2 cell line was purchased from American Type Culture Collection ( ACTT ) ( VA, USA ) . Dulbecco ‘s Modified Eagle Medium ( DMEM ) , 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 albumen ( BSA ) was from Santa Cruz Biotechnology ( CA, USA ) . MTT check ( 3- ( 4,5-Dimethylthiazol-2-yl ) -2,5-diphenyltetrazolium bromide check ) was purchased from Molecular Probes ( Oregon, USA ) . LDH check ( Lactate dehydrogenase check ) was from ROCHE ( Mannhein, Germany ) . BODIPY @ 493/503 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 class of analysis.
Culture of HepG2 cells
HepG2 cells were cultured in Dulbecco ‘s modified Eagle ‘s medium, incorporating 10 % ( v/v ) foetal bovine serum and 1 % ( v/v ) Penicillin-Streptomycin under 5 % CO2, 95 % humidness at 37 & A ; deg ; C. The cells were subcultured by utilizing 0.5 % Trypsin-EDTA 1x ( Invitrogen Corporation, NY, USA ) for withdrawal and seeded at proper cell figure in all experiments.
Fatty acid intervention
When 80 % confluency of HepG2 was reached, it was treated with assorted concentrations of the fatty acids ( 0 millimeter, 0.1 millimeter, 0.2 millimeter, 0.3 millimeter, 0.5 millimeter, 0.7 millimeter and 1.0 millimeter ) for 24 h. The stock solution of fatty acids was prepared at 100 millimeter by fade outing in DMSO and stored at -200C. The stocks were diluted in DMEM media incorporating a changeless ratio of fatty acid edge bovine serum albumen at 2 to 1 to obtain working solution in all experiments.
Cytotoxicity was based on the measuring of cytoplasmatic enzyme activity by utilizing cytotoxicity sensing kit ( ROCHE, Mannhein, Germany ) . The cytoplasmatic enzyme was released from damaged cells that its enzyme activity expresses to the proportion of toxiced-cell. Lactate dehydrogenase ( LDH ) nowadayss in all cells which is a stable cytoplasmatic enzyme. When the membrane unity of the cells is damaged, it is rapidly released into the media. In this check, NAD+ is reduced to NADH/H+ during transition of lactate to pyruvate by the LDH-catalyzed. After that, H/H+ from NADH/H+ was transferred by the accelerator ( diaphorase ) to the tetrazolium salt ( yellow ) which was reduced to formazan ( ruddy ) . To carry on the check, the civilization supernatant is collected cell-free after desire exposure clip ( 24 H ) . The reaction mixture from the kit was so applied in the samples. The soaking up of the formazan dye formed was measured at 490 nanometer on an ELISA reader ( VERSARMAX, Molecular Divices. , CA, USA ) .
Cell viability was measured based on measurement soaking up of a non-water-soluble purple formazan which was reduced from a xanthous water-soluble tetrazolium salt in unrecorded 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 fade out the furmazan crystals. After gently pipetting, the absorbency was measured at 570 nanometer utilizing an ELISA reader ( VERSARMAX, Molecular Divices. , CA, USA ) . The appraisal of cell viability was calculated by comparing between the spectra value of treated and untreated cells.
Quantification of triglyceride
Triglyceride content ( TG ) was determined harmonizing to an enzymic method ( BioVion Inc, CA, USA ) . In this method, glycerin is a merchandise by TG-catalyzed which reacts with the investigation to bring forth colour measured on spectrophotometry at 570 nanometer. In briefly, the cells were washed twice times with cold PBS, so homogenized in 5 % Triton-X100 solution. After easy heating at 80-100 & A ; deg ; C for 5 min, the samples were centrifuged at 12000 revolutions per minute for 5 min. The supernatant collected from taking indissoluble stuffs was added 2 ?l of lipase, assorted good and incubated for 20 min at room temperature. Finally, 50 ?l of the reaction mix was putted in each sample for 45 min of incubation, protected from visible radiation. The value of triglyceride content was quantified based on triglyceride criterion curve that was constructed with different concentrations of TG ( 0, 20, 40, 60, 80, and 100 nmol/ml ) .
Measurement of reactive O species ( ROS ) coevals
The measuring of ROS production within cells was carried out by utilizing 2?,7?-Dichlorohydrofluorescein diacetate ( Carboxyl-H2DCFDA ; Invitrogen Corporation, Oregon, USA ) which is combined into fluorescent merchandises in the presence of H2O2 and other ROS molecules and esterases ( Zhenyuan Song et Al, 2007 ) . After the cells were overloaded with 1.0 millimeters fatty acids, 10 mM concluding 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 200i?l RIPA buffer ( PIERCE, IL, USA ) . The lysed-cells were centrifuged at 12000 revolutions per minute for 5 min. The supernatants were conveyed to a 96-well dorsum home base which were excited at 485 nanometers and emitted at 530 nanometer for the Carboxyl-H2DCFDA fluorescence on Fluorometer ( VICTOR2, Perkin Elmer. , MA, USA ) .
Trilyceride staining on Confocal
Bodipy @ 493/503 ( Invitrogen, Oregon, USA ) was used to capture TG fluorescence on Confocal microscopy. In this experiment, the cells were prepared as above. Before the dyes intervention, the cells were washed with PBS twice times. Bodipy @ 493/503 was so added at 1.0 i?M, and 15 min of incubation at 370C after the cells were rinsed with PBS once more. Zeiss LSM Image Brown package ( LSM 510 meta, Carl Zeiss. , Jena, Germany ) was handled to take TG image at excitement of 488 and emanation of BP 505-530 nanometer.
ROS staining on Confocal
ROS coevals in HepG2 was stained by utilizing Carboxyl-H2DCFDA. In this experiment, the cells were prepared as above. Before the dyes intervention, the cells were washed with warmed PBS twice times. The carboxyl-H2DCFDA was applied at 10 mM concluding concentration in Serum free media ( DMEM without FBS ) , and incubated for 30 min at 370C, protected from the visible radiation. After that, the cells were rinsed with warmed PBS once more. Zeiss LSM Image Brown package ( LSM 510 meta, Carl Zeiss. , Jena, Germany ) was handled to take ROS image at excitement of 488 nanometers and emanation of LP 530 nanometer.
Detection of cell decease and trilyceride accretion by Confocal
HepG2 seeded in the 24-well home base and treated with concluding concentration of fatty acids to 1.0 millimeter for 24 h. After the incubation clip, the cells were washed twice times with PBS. Then, Bodipy @ 493/503 ( Invitrogen, Oregon, USA ) was dissolved in PBS at 5 i?g/ml which was added into each well. This procedure was kept in darkness for 15 min at 370C. After that, the Bodipy solution was removed and the cells were so washed by Adhering buffer from Annexin V Floustaining kit ( Roche, IN, USA ) . Finally, the cells were incubated in 100 i?l/ml of Propidium iodide ( PI ) for 10 min in darkness. Exposition of TG accretion and programmed cell death was observed at excitement of 488 and 543 nanometer, and emanation of BP 505-530 and LP 650 nanometer on Carl Zeiss Confocal Microscopy ( LSM 510 meta, Carl Zeiss. , Jena, Germany ) , severally.
All consequences were expressed as mean of repeated three or four values ± SEM. The difference between groups was identified by utilizing t.test. P & A ; lt ; 0.05 was considered statistical important.