Question

1. Describe the mechanism of DCFDA and, what is the purpose of running assay

1. 2 How will we evaluate the cytotoxicity of the 3 compounds we are working with?Must describe the mechanism of CCK-8.

Answer 1

DCFDA

Cellular ROS levels can be measured in live cells by a technique that converts 2’,7’ –dichlorofluorescin diacetate (DCFDA) which is oxidized to a fluorescence dye 2’, 7’ –dichlorofluorescein (DCF). The fluorescence generated is directly proportional to the amount of oxidized DCFDA to DCF.

Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are continually produced during metabolic processes. ROS generation is normally counterbalanced by the action of antioxidant enzymes and other redox molecules. However, excess ROS can lead to cellular injury in the form of damaged DNA, lipids and proteins. Excess reactive oxygen species must be promptly eliminated from the cell by a variety of antioxidant defense mechanisms. Cellular antioxidant enzymes and other redox molecules serve to counterbalance ROS generated in the cell.

Cytotoxity  

Extraction

Dried lichen material (100 g) were milled into powder and then extracted with methanol (2.5 l) in an extractor for 36 h. The extract was evaporated in a rotatory evaporator and dried by vacuum pump to afford the 7.5 g of methanolic extract.

Compound isolation

Methanolic fraction (5 g) was subjected to chromatography on silica gel (60–120 mesh) eluted with a step wise gradient of hexane-ethyl acetate (9.5:0.5, 9:1, 8.5:1.5, 8:2, 7.5:2.5, 7:3) by volume to afford a total of 500 fractions of 50 ml each. Column fractions were analyzed by TLC, and fractions with similar TLC patterns were combined to give five major sub column fractions. Column fraction-1 was further purified to give 21.5 mg of everninic acid (21.5 mg) white crystalline compound . Fraction 2 was further purified to give 16 mg of white powdered (2) Roccellic acid.

Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays. Dojindo’s highly water-soluble tetrazolium salt, WST-8, is reduced by dehydrogenase activities in cells to give a yellow-color formazan dye, which is soluble in the tissue culture media. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. The detection sensitivity of CCK-8 is higher than the other tetrazolium salts such as MTT, XTT, MTS or WST-1.