What Are Hepg2 Cells ( American Type Culture Collection ), Manassas, Usa

2234 Words Sep 21st, 2016 9 Pages
HepG2 cells (ATCC No. HB-8065) were obtained from ATCC (American type culture collection), Manassas, USA. The cells were maintained as a subconfluent monolayer in 75 cm2 culture flask using a modified DMEM medium with 10% (v/v) FBS + penicillin-streptomycin (50 Units/mL) and used for experiments two weeks post thawing from cryopreserved stock. The cells were harvested using prewarmed 0.25% Trypsin-EDTA. For cytotoxicity and MN study, the cells (1×104 cells/well) were seeded into 96 well plates and incubated at 37 °C under 5% CO2 in an incubator (Heracell 150i CO2 incubator, Thermo Scientific, Australia) for 24 h before chemical treatment.
Stock solutions of PAHs (B[a]P, Nap, Phe and Pyr) and metals (As, Cd and Pb) were prepared in DMSO and MilliQ water (18 MΩ.cm, (Merck Millipore, VIC, Australia), respectively. The working solutions prepared in DMEM + FBS (10%) medium or DMEM alone were used for MN test and Ahr CAFLUX assay, respectively. The working solutions were added to the plates reaching a final concentration of 0.5% v/v of vehicle control (DMSO or MilliQ water).
In vitro flow cytometry micronucleus test

The selected concentrations of individual MN study were 0 to 2 μM for B[a]P and Cd; 0 to 10 μM for As and 0 to 100 μM for Pb. Initially, the cytotoxicity of PAHs and metals were determined using the MTS assay. Based on their cytotoxicity to HepG2 cells, the concentrations of metals and PAHs were selected for the MN study. The treatment was carried out for 1.5 – 2…

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