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Quantitative Measurement of Cell Proliferation,,,Using the BrdU ELISA: A Comparison,,,Between Colorimetric and Chemiluminescent Detection

f DNA synthesis in standard medium (DMEM) was determined and compared with that in a medium containing 10 ng/ml cholera toxin (Sigma) for inhibition of fibroblast growth (DMEM + CT).

In a third set of experiments, we examined the advantages of a serum-free, special medium (PromoCell) for the cultivation of human umbilical vein endothelial cell (HUVEC) in comparison with the standard medium (RPMI + 10 % FCS).

In each case, 24 hours after seeding of the cells, BrdU was added to a final concentration of 1 M. After incubation for further 24 hours, DNA synthesis was assayed with the Cell Proliferation ELISA, BrdU (Roche Molecular Biochemicals) using colorimetric or chemiluminescence detection, both according to the manufacturer's instructions. Newly synthesized BrdU-DNA was determined using an ELISA reader (BioRad, 450) or a luminometer (Orion, Berthold Detection Systems).

Results

The results clearly show that, depending on the labeling conditions or the proliferation behavior of the cells, widely differing results are obtained with the two detection systems, colorimetry and chemiluminescence (Figure 1).



In the cases of the prostatic carcinoma cells lines P1/289 and P1/373 treated with hormone, growth factor, and toxin, an increase in the DNA labeling index of more than 50 % is observed in the chemiluminescence test whereas, for the same cells, the colorimetric BrdU assay reveals an increase of only about 20 %. Growth inhibition by cholera toxin in fibroblast cultures results in a significant reduction of BrdU incorporation from 100 % in the control to values of 44 % and 35 % in the presence of 10 ng/ml cholera toxin, irrespective of the detection system used.

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