20.109(F13): Mod 2 Day 7 HTS and Analysis

Introduction

Last time you set up a cell viability screen to evaluate the combination of an EGFR inhibitor and an inhibitor targeting a downstream pathway (PI3K/Akt, Ras/ErK, or STAT3). Today we will use a luminescence based assay to quantify the extent of cell death within your culture. If the robot in the Koch Institute is cooperating (oh the perils of high technology!) we will depend on it to do all the work for us! Otherwise, we will employ a 96-well multichannel pipette to significantly cut down on our work -- giving us a different method to minimize our pipetting steps.

The cell viability assay that we will use is from Promega, Inc., and is called the CellTiter-Glo assay. The CellTiter-Glo reagent contains a recombinantly produced Luciferase that catalyzes the mono-oxygenation of beetle luciferan to oxyluciferan + light. The reaction requires Mg2+ and ATP -- and the source of ATP for the reaction is from your cells. Therefore, the extent of reaction (i.e. light generation) is related to the number of cells within the culture. Promega reports that the reaction proceeds linearly within the range of 15 and > 50,000 cells. The doubling time of our SKOV3 cells is approximately 30 hrs, therefore our experiment will be safely within this range.