Culture Of Hepg2 Cells Biology Essay

Culture Of Hepg2 Cells Biology EssayHep G2 cubicle line was purchased from American emblem Culture Collection (ACTT) (VA, USA). Dulbeccos Modified Eagle Medium (DMEM), 0.5% Trypsin-EDTA 10x, and Penicillin-Streptomycin (PS) were obtained from Invitrogen Corpo symmetryn (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 ovalbumin (BSA) was from Santa Cruz Biotechnology (CA, USA). MTT hindrance (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). emailprotected/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). inorganic 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 otherwise chemicals met in standard grade of epitome.Culture of HepG2 cellsHepG2 cells were cultured in Dulbeccos modified Eagles medium, containing 10% (v/v) fetal bovine serum and 1% (v/v) Penicillin-Streptomycin under 5 % CO2, 95 % humidity at 37C. The cells were subcultured by use 0.5% Trypsin-EDTA 1x (Invitrogen Corporation, NY, USA) for detachment and seeded at proper cell number in all experiments.Fatty acid treatmentWhen 80 % confluency of HepG2 was reached, it was treated with various concentrations of the fatty acids (0 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.5 mM, 0.7 mM and 1.0 mM) for 24 h. The job 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 a constant ratio of fatty acid bound b ovine serum albumin at 2 to 1 to obtain working solution in all experiments.Cytotoxicity assayCytotoxicity 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. by and by 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 painting time (24 h). The reaction cockture from the kit was then applied in the samples. The absorption of the formazan d ye formed was measured at 490 nm on an enzyme-linked-immunosorbent serologic assay reader (VERSARMAX, Molecular Divices., CA, USA).Cell viabilityCell 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 triglycerideTriglyceride content (TG) was determined fit in 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 process in two ways times with cold PBS, then homogenized in 5% Triton-X100 solution. After slowly heating at 80-100C for 5 min, the samples were centrifuged at 12000 rpm for 5 min. The supernatant collected from removing insoluble materials was added 2 l of lipase, mixed well 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 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). step of reactive oxygen species (ROS) generationThe 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 water- wash twice times with warmed PBS and lysed in 200l 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 delirious at 485 nm and emitted at 530 nm for the Carboxyl-H2DCFDA fluorescence on Fluorometer (VICTOR2, Perkin Elmer., MA, USA).Trilyceride staining on ConfocalBodipy 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 treatment, the cells were washed with PBS twice times. Bodipy 493/503 was then added at 1.0 M, and 15 min of incubation at 370C after the cells were rinsed with PBS again. Zeiss LSM Image Brown software (LSM 510 meta, Carl Zeiss., Jena, Germany) was handled to take TG impression at excitation of 488 and emission of BP 505-530 nm.ROS staining on ConfocalROS generation in HepG2 was stained by using Carboxyl-H2DCFDA. 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 10 mM final concentration in Serum free media (DMEM without FBS), and incubated for 30 min at 370C, protected from the light. After that, 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 image at excitation of 488 nm and emission of LP 530 nm.Detection of cell death and trilyceride accumulation by ConfocalHepG2 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 g/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 l/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.Data analysisAll results were expressed as mean of repeated three or four values SEM. The difference between groups was identified by using t.test. p 0.05 was considered statistical significant.