In recent years there has been a significant change in the way toxicity testing of test components is conducted. In general, the emphasis has changed from in vivo animal methods to in vitro toxicity methods. Following the introduction of in vitro assays, several groups have assessed the efficiency of testing chemicals in vitro. (1)
One such assay is the Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. This assay has several advantages that include ease of use, precision, and rapid determination of toxicity. (8)
Typically, in MTS-based assays, toxicity is determined using the dose-response curve to determine IC50, the concentration of the test substance required to reduce the light absorbance capacity of exposed cell cultures by 50%. However, IC50 values can also be determined by generating a standard curve consisting of a specified range of cell dilutions, because a 50% reduction in absorbance may not equate with a 50% reduction in cellular viability. This study investigated whether either method for determining IC50 affects the accuracy of the assay.
Materials and Methods
Test chemicals were selected by number from different categories in the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) list (2)
. Chemicals selected include: glycerol, malonic acid, nicotine, phenol, potassium hydroxide, sodium dichromate and sodium hydroxide. HeLa cell line (ATCC) was selected for this study due to its consistent growth and ease of maintenance. The cells were grown in 75cm2 tissue culture flasks. The culture growth medium consisted of a 1:1 ratio of DMEM (Dulbecco’s Modified Eagle’s Medium; Sigma) and RPMI-1640 (Sigma), supplemented with 10% newborn bovine serum (Trace Bioscience) and L-glutamine (1mM), penicillin (50U/ml) and streptomycin (0.05mg/ml) (Sigma).
The CellTiter 96® AQueous Assay uses the novel tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) and the electron coupling reagent, phenazine methosulfate (PMS). MTS is chemically reduced by cells into formazan, which is soluble in tissue culture medium. (8)
The measurement of the absorbance of the formazan can be carried out using 96 well microplates at 492nm. The assay measures dehydrogenase enzyme activity found in metabolically active cells.
Since the production of formazan is proportional to the number of living cells, the intensity of the produced color is a good indication of the viability of the cells. MTS solutions were prepared according to the manufacturer’s instructions. (9)
Stock PMS (Sigma) was dissolved in PBS at a concentration of 0.92mg/ml DPBS (0.92mg/ml PMS in DPBS is also included with the CellTiter 96® AQueous Assay System from Promega). The solutions were then stored in light-protected tubes at -20°C. MTS and PMS detection reagents were mixed, using a ratio of 20:1 (MTS:PMS), immediately prior to addition to the cell culture at a ratio of 1:5 (detection reagents:cell culture medium).
Chemical Treatment of Cells
To prepare stock solutions, usually as 10% w/v solutions, the chemicals were dissolved directly in culture medium. The stock solutions were filter-sterilized (0.22µm). The resultant solutions were kept at 4°C and used within 24 hours for the assay. Five separate serial dilutions of each test chemical in culture medium were prepared for addition to cells.
HeLa cell suspensions were prepared in a standard manner. (10)
Culture medium was removed from the flask and the cells were rinsed three times with DMEM. Approximately 5ml of Trypsin EDTA solution (Trace Bioscience) were then added to the flask and incubated at 37°C for a few minutes. Cells were washed three times with fresh medium and resuspended at a concentration of 2 x 106 cells/ml. Only cells with viability greater than 95%, as determined by Trypan Blue dye exclusion, were used for testing.(9)
The cell suspension was then added to each dilution of the test chemical at a ratio of 1:9 (cell suspension:test chemical solution).
The same series of dilutions was prepared without addition of cells as background control samples. These controls are essential for toxicity testing, as generation of 492nm absorbance often occurs, especially when high concentrations of test chemicals are added to the MTS and PMS assay mixture. (8)
A sample of culture medium was used as a "medium-only" control (IC0). The same cell suspension as that prepared for the assay was also used as a "cell-only" control (IC100).
The final solutions were added, in four replicates, into a 96 well microplate (100µl in each well) using a multichannel pipettor. The microplate was then incubated in a humidified, 5% CO2 incubator at 37°C for 20 hours. The detection reagent was pipetted into each well of the microplate at a ratio of 1:5 (detection reagent:content of each well of microplate) (Figure 1). The microplate was incubated for another 4 hours under identical conditions.
Figure 1. Depiction of test sample layout of a 96 microwell plate.
IC50 Determination by the Conventional Method
Background absorbance, due solely to the reaction of the reagents and each test chemical, was deducted from the absorbance values generated by the exposed cells. The level of background, in general, increases with the amount of test compound used in the assay. The mean absorbance (of four replicates) generated by the "medium-only" control is denoted as IC0. The mean absorbance generated by the "cell-only" control is denoted as IC100.
To determine the IC50 value, the IC50 absorbance value was first calculated using the following formula:
||(AbsIC100 + AbsIC0)|
Then, the X-axis intercept of the dose-response curve at the point determined by the above formula was considered as IC50 value for each test chemical (Figure 2).
Figure 2. Conventional method of determining IC50 using the dose-response curve.
IC50 Determination by the Standard Curve Method
A standard curve for HeLa cells was prepared for each set of experiments using the same solution of cells. A microplate containing 40 x 103 cells/ well was prepared. Then, five dilutions (20, 10, 5, 2.5 and 1.25 [ x 103cells/well]) were prepared from the original 40 x 103 cells/well. Cells were seeded by transferring the cell dilutions into the appropriate well of the microplate. Each dilution was performed in four replicates.
The absorbance values were recorded after incubating for 20 hours as described above. We performed the cell dilution plate alongside the exposed cells to generate the standard curve. Therefore, the exposed cells (for which we have defined a cell number) and cell dilution plate forming the standard curve underwent the same incubation conditions. In addition, we performed the experiments using cell numbers that only fall in the linear range of the standard curve to reduce error. A typical standard curve is shown in Figure 3.
Figure 3. Standard curve of HeLa cell absorbance at 492nm versus cell concentration.
To determine the IC50 value using the standard curve method, a reading of the absorbance representing 104 cells (50% of the total population in each well) was specified. This absorbance value was then used to determine the IC50 value using the X-axis intercept of the dose-response curve, as described in the conventional method above.
The IC50 value for each test chemical was determined using three replicates of each experiment. A comparison of the IC50 results for the conventional method versus the standard curve method is shown in Figure 4, and a comparison between the two IC50 determination methods is shown in Figure 5.
Figure 4. A comparison between IC50 values calculated by absorbance and cell number (1, glycerol; 2, malonic acid; 3, nicotine; 4, phenol; 5, potassium hydroxide; 6, sodium dichromate; 7, sodium hydroxide).
Figure 5. Comparison between two methods of IC50 determination.
A ranking analysis was also conducted. The results of the analysis from both methods were identical, and in order of increasing toxicity, the chemicals ranked as follows: glycerol < phenol < nicotine < malonic acid < potassium hydroxide < sodium hydroxide < sodium dichromate. There was no significant difference between the conventional and standard curve methods using the student's t-test (p=0.12).
In the conventional method of determining IC50 values, it is assumed that absorbance values are directly and precisely proportional to cell numbers. Therefore, as seen in Figure 2, it is presumed that absorbance value generated by the formula represents the absorbance value for 50% of the intact cells.
However, as evident in Figures 3 and 5, this is not the case. In fact, the absorbance value generated by 50% of intact cells according to the standard curve is greater than what is obtained using the formula. As a result, if the standard curve method of determining the IC50 absorbance value is considered, a lower value will be achieved. Therefore, it is concluded that the standard curve method generates a more accurate IC50 value.
The authors thank Dr. R. Rosen for his critical comments on this manuscript.