COURSE CODE: MBTE 4034
COURSE NAME: Methods in Molecular Biotechnology Lab II
STUDENT NAME: Kai Wen Cheng
DATE OF EXPERIMENT: 15th February, 2017
Cytotoxicity assay is a powerful tool in the biotechnological field, especially during the process of drug development. The application of cytotoxicity in the pharmaceutical industries produces data of killing ability of the drugs, in advance of the releasing to the public. Precisely, the assay measures the LD50, the concentration of potent toxicant leading to the 50% reduction of the absorbance in comparison with the control group, without treating with tested chemical, with the absorbance be determined spectrophotometrically with the reference wavelengths of 550 nm and 660 nm respectively. (“Cell Viability”) (“A Simple Method To Measure Cell Viability In Proliferation And Cytotoxicity Assays”)
In the experiment, the cytotoxicity assay is to be conducted on the mammalian cells cultured in advance, with the purpose of investigating the potential toxic effect of staurosporine. The staurosporine will be diluted with DMSO, as the solvent, then added with different concentration to the 96-well culture plate carrying the said mammalian cells. The aim of the experiment is to testify whether higher concentration of staurosporine leads to the higher cytotoxicity rate.
For measuring the cytotoxicity, the MTT solution will be added to each wall of the 96-well culture plate. MTT can be reduced by succinate dehydrogenase in the mitochondria, result in the conversion of yellow salts to blue-colored formazan crystals.
As the result of the experiment, a conclusion can be made that higher the concentration of staurosporine added, higher the cytotoxicity. (“Measuring Cell Viability / Cytotoxicity”)
The procedure was as described in Experiment Animal #2 of the manual of Methods in Molecular Biotechnology Lab II.
The experiment was conducted to test the cytotoxicity of the chemical staurosporine. After the cytotoxicity assay, the following results were obtained.
OD of the cells
The dilution of staurosporine was done as the following table:
Noted that for tubes V to II, the concentration of DMSO were approximately to be close to 0, therefore were omitted. The diluted solutions were added to the 96-well culture plate according to the following distribution:
Noted that the tubes VI to IX from the lanes 6, 7 and 8 containing DMSO only, whilst the tubes II to IX from the lanes 101 11 and 12 were containing staurosporine.
After several laboratorial procedures, the OD value were measured as the followings:
Calculation of the mean
Before the further computing, the average value of each biological replicates were required to be calculated. The average value of OD was obtained as following:
With the OD value of the control as 3.455, using the equation for calculating the cytotoxicity, (OD of control-OD of sample)/OD of sample, we could plot a dose-response curve as following:
The correlation of the concentration of staurosporine with the cytotoxicity rate is indicated with the trend line drawn.
Calculated from the equation shown at the bottom right of the curve, the LD50 value would be 3.4%, meaning that the concentration of 3.4% of staurosporine would kill 50% of the cells.
Comparison of the cytotoxicities of staurosporine and DMSO
The comparison of the cytotoxicities of staurosporine and DMSO could be shown in the following bar chart:
Perhaps due to the mistakes on manipulation during the experimental process, there was an abnormal bulge shown between 0 to 0.01 staurosporine concentration in Figure. 1. However, the correlation of the concentration of staurosporine and the cytotoxicity were still considered to be obvious when seeing from the curve plotted.
From Figure. 2, it was clear that the cytotoxicity of staurosporine was generally higher than DMSO. The trend of the higher concentration leading to higher cytotoxicity was not clear in the case of DMSO, however, comparing the relatively higher cytotoxicity in VIII & IX then to VI & VII, the assumption of the higher concentration of the chemical leading to higher cytotoxicity could be possible. As for the case of the staurosporine, the trend of the higher concentration of the chemical leading to higher cytotoxicity was obvious, as discussed before. (“Determining The Predictive Mechanism Of Toxicity Using A Single-Well Multiplexed Assay With The Apotox-Glo Triplex Assay”)
As the conclusion, the hypothesis of the higher concentration of staurosporine lead to the higher cytotoxicity rate was not rejected.
“A Simple Method To Measure Cell Viability In Proliferation And Cytotoxicity Assays”. Histology. N.p., 2017. Web. 1 Mar. 2017.
“Cell Viability”. Worldwide.promega.com. N.p., 2017. Web. 1 Mar. 2017.
“Determining The Predictive Mechanism Of Toxicity Using A Single-Well Multiplexed Assay With The Apotox-Glo Triplex Assay”. Worldwide.promega.com. N.p., 2017. Web. 1 Mar. 2017.
“Measuring Cell Viability / Cytotoxicity”. N.p., 2017. Web. 1 Mar. 2017.