( 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 medium without cells in replicates of 5. Swirl the plate to evenly distribute cells in the wells. Incubate plate overnight at 37C.

4. Prepare XTT labeling mix according to instructions and add 50 l to each well. Incubate at 37C for 1, 2, and 3 hr.

5. After 1 hr of incubation, place the plate into the Benchmark Plus.

6. To determine the optimum wavelength for the assay, run a spectral scan. In Microplate Manager PC software, select File>New Endpoint Protocol>Advanced Options>Scan Well. Scan three wells with different cell densities a cross the wavelength range 340800 nm with a step size of 5 nm. Next, construct a graph to show the peak wavelength around 475 nm (the re commended wavelength for reading the assay) for all samples scanned.

7. After the spectral scan, open the Microplate Manager software application. Under the File menu, select New Endpoint Protocol. Next, select the 96-well format and edit the template, selecting the wells used. Specify a single-wavelength reading at the peak wavelength determined in step 6.

8. Assay the plate using the Benchmark Plus by clicking the Run button. Repeat for the 2 and 3 hr incubation times.

9. After reading the samples, export or copy and paste the data points into Microsoft Excel software and plot them.

Results
Figure 1 shows the results obtained by scanning sample wells with different cell densities (step 6 of Procedure). The scan resulted in an absorbance curve that peaked at about 475 nm, the wavelength specified for sample reading in the XTT kit instructions.

The assay was performed at 475 nm. Figure 2 shows a proportional increase in the OD value at 475 nm with increasing cell number.

Conclusion
The measurement of cell viability and cell division is used in a wide range of experiments to determine growth factor activity, for drug screening, and to determine the effects of chemical transfectants on human cells. We tested the Roche cell proliferation kit for measurement of cell density of A549 cells grown in 96-well plates. After 24 hr, when the cells were ~90% confluent in the wells with the most cells, we performed the cell proliferation assay.

At low cell density (cells ~50% confluent on the plate), there was a proportional increase in OD 475 with increasing cell number. At high cell density, the increase in OD 475 was not proportional to the increase in cell number. As seen in the figure, this increase was independent of the length of incubation with the reagent between 1 and 3 hr. However, at shorter incubation times (0.5 hr, data not shown), the OD 475 was proportional to cell number up to 5070% confluence.

Bibliography
Guide to Cell Proliferation and Apoptosis Methods, Roche Applied Science, available online at http://www.roche-applied-science.com/fst/publications.htm?/ prod_inf/manuals/cell_man/cell_toc.html

Roehm NW et al., An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT, J Immunol Methods 142, 257265 (1991)

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