( William C Heiser PhD an...)Cell Proliferation Assay Using the Benchmark Plus Microplate Spectrophotometer

William C Heiser, PhD and Ma Theresa Redila-Flores, Bio-Rad Laboratories, Inc . , Hercules, CA 94547 USA

Introduction
An accurate and rapid assessment of viable cell number and proliferation is often required for in vitro and in vivo experiments. These assessments are necessary and useful for analysis of growth factor activity, drug screening, serum batch testing, and toxicological determination of carcinogenic compounds, to name a few. Cell proliferation kits are used to measure cell viability and division in response to growth factors, cytokines, and nutrients, as well as growth inhibition by cytotoxic agents. The cell proliferation kit described here utilizes a microplate spectrophotometer to quantitate an assay based on XTT, a tetrazolium salt that is cleaved as a result of metabolic activity to generate a colored formazan product.

Method
A cell proliferation kit from Roche was used to measure proliferation of A549 cells grown at different densities overnight. The cells were incubated with XTT before measuring the absorbance. To confirm the optimal wavelength at which to read the samples, a spectral scan was done. The cells were then read after 1, 2, and 3 hr of incubation as suggested in the kit instructions.

Procedure
1. Inoculate A549 cells into a 25 cm² flask with DMEM or appropriate medium. Grow cells to 70% confluence.

2. Wash cells with 1x PBS. Trypsinize, then add 2 ml of appropriate medium.

3. Dilute cells 10-fold, to about 2.5 x 10⁵ cells/ml. Prepare 2-fold dilutions and plate 100 l of each dilution in replicates of 5 in a 96-well plate. Prepare a blank by dispensing
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