Abstract:

We evaluated the cytotoxicity of 5-methyl chrysene in the FE1 murine lung epithelial cells. Cytotoxicity was determined using the sulphorhodamine B assay (SRB) at 3 time points post-exposure (24, 48, and 72 hours) and 5 treatment groups. The treatment groups were a control, solvent control (DMSO), and 5-methyl chrysene at 500 pM, 1 nM, 500 nM, and 1 uM (final concentrations). The assays were carried out in 96-well microplates with 16 technical replicates per treatment group. SRB fluorescence was measured on a Tecan microplate reader following the published SRB assay.

Suggested Citation:

Jeffrey Wickliffe. 2017. Cytotoxicity of 5-methyl Chrysene in FE1 Murine Lung Epithelial Cells. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N789148M

Purpose:

The purpose of this experiment was to determine the concentration-response relationship between the aklylated PAH, 5-methyl Chrysene and defined concentrations spanning 2 orders of magnitude, and toxicity in the FE1 murine lung epithelial cell line, in vitro. Toxicity was determined using the SRB assay.

Data Parameters and Units:

The concentrations of 5-methyl Chrysene (5mC) are reported in final molar concentrations (nanomolar-nM to micromolar-uM) in cell culture medium (serum-free) and range from 50 nM to 1,000 nM with additional test concentrations at 100 nM, and 500 nM. The amino acid binding dye sulphorhodamine b is used as the indicator for measuring cytotoxicity. The dye is fluorescent so the units are in arbitrary fluorescence values. We also normalize all of our data to % toxicity of the control (untreated) cells. Date (MM/DD/YYYY), Time (HH:MM:SS), Excitation wavelength (nm), Emission wavelength (nm), Excitation bandwidth (nm), Emission bandwidth (nm), Integration time (us), Lag time (us), Settle time (ms), Z-position (um), Temperature (deg C), Timepoint (hrs)

Methods:

Data from 96-well microplates used for cytotoxicity as measured by SRB fluorescence is arrayed in a 12 x 8 format. Wells in the microplates range from A1 to H12 with letter designations A-H corresponding to rows and numerical designations (1-12) corresponding to columns. Columns 1 and 2 are untreated controls, columns 3 and 4 are solvent-only (DMSO) treated controls, columns 5 and 6 are treated with 50 nM 5mC, columns 7 and 8 are treated with 100 nM 5mC, columns 9 and 10 are treated with 500 nM 5mC, and columns 11 and 12 are treated with 1,000 nM or 1 uM 5mC. Cytotoxicity was assayed at 24, 48, and 72 hours post-exposure in which cells were treated for 4 in serum-free medium. Treatment medium was then removed and replaced with fresh complete, culture medium. Data were then aggregated by way of computing averages and standard deviations for each treatment group across the respective 16 technical replicates. Data were then further processed to normalize responses (i.e. toxicity) to the untreated controls effectively designating untreated controls as 100% viable.

Instruments:

Tecan microplate reader used for assessing SRB fluorescence in wells of experimental microplates.

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