Resumen de In Vitro Characterization of Dopaminergic Toxins: A Laboratory Exercise Suitable for a Senior Undergraduate Biochemistry Laboratory Curriculum
Viet Le, Joseph Adjei, Maya Eldani, Adetuyi Oluwatosin, Kandatege Wimalasena
The objective of this study was to introduce a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay and a fluorometric 4′,6-diamidino-2-phenylindole (DAPI) apoptotic assay that are suitable to adopt into an undergraduate biochemistry laboratory curriculum to identify and characterize dopaminergic toxins in the common working environments. Here, we used commercially available Parkinsonian toxin, 1-methyl-4-phenylpyridinium (MPP+), as a model dopaminergic toxin and tested its selective dopaminergic toxicity in MN9D cells against non-neuronal HepG2 cells in an upper-level undergraduate biochemistry laboratory class. Overall, this laboratory exercise helped students to develop skills in quantitative in vitro toxicity measurements using a simple colorimetric MTT assay and gain an understanding of the mechanism of toxicity by DAPI fluorescence imaging techniques for apoptosis. Required experimental data analyses will also allow students to gain an understanding of the use of appropriate statistical analyses of biochemicals in biomedical studies. More importantly, this exercise will allow students to better understand the intimate relationship between the environment and human health. The high relevance of this study to environmental effects of human health will certainly result in high interest leading to increased student engagement and interest for advanced learniThe objective of this study was to introduce a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay and a fluorometric 4′,6-diamidino-2-phenylindole (DAPI) apoptotic assay that are suitable to adopt into an undergraduate biochemistry laboratory curriculum to identify and characterize dopaminergic toxins in the common working environments. Here, we used commercially available Parkinsonian toxin, 1-methyl-4-phenylpyridinium (MPP+), as a model dopaminergic toxin and tested its selective dopaminergic toxicity in MN9D cells against non-neuronal HepG2 cells in an upper-level undergraduate biochemistry laboratory class. Overall, this laboratory exercise helped students to develop skills in quantitative in vitro toxicity measurements using a simple colorimetric MTT assay and gain an understanding of the mechanism of toxicity by DAPI fluorescence imaging techniques for apoptosis. Required experimental data analyses will also allow students to gain an understanding of the use of appropriate statistical analyses of biochemicals in biomedical studies. More importantly, this exercise will allow students to better understand the intimate relationship between the environment and human health. The high relevance of this study to environmental effects of human health will certainly result in high interest leading to increased student engagement and interest for advanced learning.
