Abstract:

The dataset consists of a single excel file that contains the % cell viability after 24-hour exposure to various concentrations of chemicals. The starting concentration of cells was 1*10‌^3 cells per well. A modified version of the trypan blue dye exclusion test was used to determine cytotoxicity tests.

Suggested Citation:

Ahmad Alqassim, Charles Miller. 2019. The trypan blue cytotoxicity assay in the human hepatocarcinoma cell line (HepG2). Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-9e9p-xw65

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 gene expression experiments.

Data Parameters and Units:

The dataset includes - treatment; and % cell viability. The percent of blue (dead) cells was determined after manually counting at least 200 cells under 200x magnification using an inverted microscope. The treatment refers to the addition of polycyclic aromatic hydrocarbons (PAHs) at a known concentration. A list of PAH tested is included in the dataset.

Methods:

HepG2 cells were used to test crude oil, oil fractions, and PAHs. HepG2 cells were incubated in a Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 μg/mL streptomycin, 50 U/mL penicillin and propagated in an incubator with 5% CO2 at 37°C. Cytotoxicity tests for determining the dose-response to acute exposure (24 h) to chemicals was a modified trypan blue dye exclusion test (Strober, 2001). The assay was carried out using 24-well plates. Cells were plated at a concentration 1 × 10^3^ cells per well. After allowing cells to adhere for 24 h, chemicals were added at concentrations of 10, 5, or 2.5 µM using a DMSO vehicle. The final DMSO concentration did not exceed 0.5%. The next day, the cell culture medium was removed and replaced with 100 µl of phosphate buffered saline (PBS) and an equal volume of a 0.4% trypan blue solution in PBS was added. After 2-3 minutes the solution was removed and replaced with PBS alone. The percent of blue (dead) cells was determined after manually counting at least 200 cells under 200x magnification using an inverted microscope according to the equation below. % Viable cells = (1.00 – (Number of blue cells ÷ Number of total cells)) × 100.

Instruments:

Inverted microscope

Provenance and Historical References:

Strober, W. (2001). Trypan Blue Exclusion Test of Cell Viability. Current Protocols in Immunology. doi:10.1002/0471142735.ima03bs21

Individual Files: