Methods for determining the proliferation of cells in response to EGFR ligands

The evaluation of cell proliferation can be accomplished by several methods. The number of cells can be determined directly by counting manually (e.g., hemocytometer) or automatically (e.g., Coulter counter or flow cytometer). The amount of DNA, which reflects the number of cells or the stage of the cell cycle, can be quantified by incorporation of labeled nucleotides (e.g., [3H]-thymidine) or nucleic acid stains (e.g., propidium iodide). Alternatively, the relative metabolic activity, which is correlative with the number of cells, can be determined through the use of metabolic dyes and measurement of the colored metabolites (e.g., MTT and MTS). Each assay has its advantages and limitations. Determining which assay to use will depend on the equipment available, the experimental design, and the questions being addressed. In this chapter we will describe methods for the use of a hemocytometer, [3H]-thymidine incorporation, cell cycle analysis with propidium iodide by flow cytometry, and evaluation of cellular metabolic activity with the MTS reagent.