Abstract:

The human hepatocarcinoma cell line (HepG2) was used to define the toxicity of chrysenes and other references polycyclic aromatic hydrocarbons (PAHs) in a series of assays where HepG2 cells were exposed to various concentrations of PAHs for 48 hours in Dulbecco’s modified Eagle’s medium.

Suggested Citation:

Ahmad Alqassim, Charles Miller. 2019. The sulforhodamine B (SRB) Cytotoxicity assay in the human hepatocarcinoma cell line (HepG2). Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-narw-9305

Publications:

Alqassim, A. Y., Wilson, M. J., Wickliffe, J. K., Pangeni, D., Overton, E. B., & Miller, C. A. (2019). Aryl hydrocarbon receptor signaling, toxicity, and gene expression responses to mono‐methylchrysenes. Environmental Toxicology, 34(9), 992–1000. doi:10.1002/tox.22770

Purpose:

To use the sublethal concentrations in the gene expression experiments.

Data Parameters and Units:

Treatment; % cell viability. Treatment refers to exposure to a particular compound and a concentration. The information on the different compound and their concentration used for the treatment are provided in the dataset.

Methods:

HepG2 cells were maintained in a Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 μg/ml streptomycin, 50 U/ml penicillin, 2 mmol/l L- glutamine, and nonessential amino acids in a humidified incubator with air plus 5% CO2 at 37°C (Lopez- Terada et al., 1986). To determine the dose-response to extended chemical exposure, the sulforhodamine B (SRB) assay was used according to Vichai and Kirtikara (2006). The SRB assay was conducted in 24-well plates under dim lights to minimize photoactivation of PAHs. HepG2 cells were seeded at 1 × 10^4^ cells per well in 1 mL DMEM as described above. Cells were allowed to proliferate for 4 days in the presence of 5, 0.5, 0.05, or 0.005 µM of the chemicals. DMSO (vehicle) was at a final concentration of 0.05% or less for these experiments. The amount of SRB dye bound was calculated using the fluorometric function of Tecan Infinite M200 Pro™ at excitation and emission wavelengths of 488 and 585 nm, respectively. To determine the cytotoxicity during extended chemical exposure, the sulforhodamine B (SRB) assay was used. The SRB assay was conducted in 24-well plates under dim lights to minimize photoactivation of PAHs. Cells were seeded at 1 × 104 cells per well in 1 mL DMEM as described above. Cells were allowed to proliferate for 4 days in the presence of different concentrations of the chemicals. The cytotoxicity assays were repeated three times for each experiment conducted in this study. DMSO (vehicle) was always at a final concentration of 0.5% or less for these experiments. The amount of SRB dye bound was determined by fluorometry. Data were reported as percent cell density of treated cells relative to the untreated, control cells at 96 h post- treatment using the equation below. % Cell density = (mean dye OD sample – mean OD background) ÷ mean OD negative control x 100. More details are provided in Vichai and Kirtikara (2006).

Instruments:

The fluorometric function of Tecan Infinite M200 Pro™ at excitation and emission wavelengths of 488 and 585 nm, respectively.

Provenance and Historical References:

Vichai V, Kirtikara K. (2006). Sulforhodamine B colorimetric assay for cytotoxicity screening. Nature protocols, 1(3), 1112-1116. doi: 10.1038/nprot.2006.179. López-Terrada D, Cheung SW, Finegold MJ, Knowles BB. (1986). Hep G2 is a hepatoblastoma-derived cell line. cancer, 4, 129-39. doi: 10.1016/j.humpath.2009.07.003.

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