Suitability of cell metabolic colorimetric assays for assessment of CD4+ T cell proliferation: comparison to 5-bromo-2-deoxyuridine (BrdU) ELISA

Abstract

The conventional tritiated thymidine ( 3 H -TdR) incorporation assay is considered as the `gold standard' for the assessment of cell growth. However, the 3 H -TdR incorporation assay has several disadvantages which have prompted the development of nonradiolabelling proliferation assays such as 5-bromo-2-deoxyuridine (BrdU) ELISA, tetrazolium microplate assay and acid phosphatase assay. In studies, these three proliferation assays have shown equivalent sensitivity and reproducibility to the 3 H -TdR incorporation assay. However the results may be affected by the cell type studied. In the present study, we have used these three proliferation assays for the assessment of rat lymph node CD4+ T lymphocyte growth in response to polyclonal antigen stimulation. The proliferation assays were compared on the basis of four criteria: sensitivity, reproducibility, stimulation index and insensitivity of the assay to the cell number. Our results indicated that the BrdU ELISA demonstrated the highest sensitivity, reproducibility and stimulation index but had a limited linear range for cellular growth. The tetrazolium microplate assay also had a relatively good sensitivity, reproducibility, stimulation index and a wider linear response range for cell growth in comparison to the BrdU ELISA. The acid phosphatase assay showed the lowest reproducibility and stimulation index. Because BrdU incorporation in DNA of proliferating cells has been reported to block cell division, we have investigated this possibility in sequential assays. Our results indicated that in our experimental conditions no evidence of an anti-mitogenic action of BrdU was observed. We also compared the performance of the MTS assay and BrdU ELISA in measuring substance P-induced CD4+ T cell proliferation. The results indicated that the MTS assay may reflect change in cell activation leading to an overestimate of cell growth. In conclusion, our results indicate that the BrdU ELISA is the most sensitive of the three proliferation assays used for the assessment of CD4+ T lymphocyte growth and is the preferred assay when small changes in cell growth are expected.