Live/Dead Assay

Abstract

LIVE/DEAD Viability/Cytotoxicity assay was conducted to see the effects every compound we used would have on the cell, whether it would help it proliferate or killed them instead. The way the assay works is that it has two different mechanisms that will produce fluorescence in the cells, green if they are alive and red if they are dead. Due that the compound used is organic; the presence of a vehicle is needed, this time the vehicle is ethanol which it is already known that it is toxic for the cells in previous experiments, so the hypothesis concluded is that ethanol will kill cells but in combination with the reagent most cells will be killed. In the obtained results it was shown that theobromine with ethanol killed more cells than the ethanol by itself.

Introduction

The compound used was theobromine, it was chosen due that it is found in chocolate and it is really easy for humans to consume it. So the main objective of the experiment was to determine what effect the reagent would have on the MCF-7 cells (a human breast cancer), would it help them to live longer, or would it kill them faster. This time the LIVE/DEAD viability/cytotoxicity assay was done, it has two mechanisms that will make the cells fluorescent depending on their state, if they are alive the color would be a bright green, and if the cells are dead a red color would be produced. The mechanisms that the assay contains are; esterase activity and plasma membrane integrity, the first one is by using calcein AM which a cell-permeant dye, it is not fluorescent until it is retained inside the cell and due to enzymatic conversion it will produce a green fluorescence in the cell. The other mechanism which is plasma membrane integrity will occur by using the nucleic acid stain Ethidium Homodimer-1, it will entered dead cells due to the fact that their membranes are damaged, it will cause no effect on healthy cells, by entering the dead cell the EthD-1 will bind to nucleic acids and will produce a red fluorescence. Due that our reagent will need a vehicle that is toxic, untreated cells will be treated alone with the vehicle to see what happens and then it can be concluded what the reagent is doing. A positive and a negative control are needed, the positive which is adding camptothecin to the cells which will induce apoptosis killing most or all of the cells, and the negative control which are untreated cells that will obviously proliferate due that there is nothing affecting them. There are tests that prove that theobromine stops the formation of tumors, but there are none stating what its effect on cancerous cells is, even though the hypothesis is that the reagent will kill more cells due to the toxicity of the vehicle and its own effect on the cells.

Methods

In the beginning first one milliliter of 2x10^5 cells/ml were added to four wells labeling as A for the negative control, B for the positive control, C for the reagent, and

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