Sharmistha Gupta1 and Mithun Mukherjee2
1West Bengal State Council of Science and Technology, West Bengal-India
2Ramakrishna Vivekananda Mission Institute of Advanced Studies, West Bengal-India
Objectives: In the present experiment a very useful cell-based high throughput screening method that uses a novel bio-sensor which specifically identifies apoptosis based on fluorescence resonance energy transfer technology.
Methodology: A stable HeLa cell line expressing a FRET-based bio-sensor protein was produced. Cells undergoing apoptosis have an activated enzyme ‘caspase-3’ which is a protease this activated enzyme breaks sensor protein and causes the fluorescence emission spectra to shift from 590 nm to 512 nm changing color from yellowish green to blue. A reduction in the green/blue emission ratio indicated apoptosis. After growing the sensor cells in 96-well plates different chemical compounds are added to the wells wherein the fluorescent profile was measured using fluorescent plate reader.
Results: It is a novel cell based HTS method and was proved to be highly effective and sensitive process for detecting anti-neoplastic compounds which induce apoptosis. In this experiment we screened compounds obtained from two plants used in Indian medicines.
Keywords: Drug discovery, HTS, FRET, fluorescent protein, apoptosis, caspase, anticancer drugs; herbal medicine, tannins, quercetins.