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

The conventional tritiated thymidine (H-3-TdR) incorporation assay is consi dered as the 'gold standard' for the assessment of cell growth. However, th e H-3-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 phosphat ase assay. In studies, these three proliferation assays have shown equivale nt sensitivity and reproducibility to the H-3-TdR incorporation assay. Howe ver the results may be affected by the cell type studied. In the present st udy, we have used these three proliferation assays for the assessment of ra t lymph node CD4 + T lymphocyte growth in response to polyclonal antigen st imulation. The proliferation assays were compared on the basis of four crit eria: sensitivity, reproducibility, stimulation index and insensitivity of the assay to the cell number. Our results indicated that the BrdU ELISA dem onstrated the highest sensitivity, reproducibility and stimulation index bu t had a limited linear range for cellular growth. The tetrazolium microplat e assay also had a relatively good sensitivity, reproducibility, stimulatio n index and a wider linear response range for cell growth in comparison to the BrdU ELISA. The acid phosphatase assay showed the lowest reproducibilit y 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 experim ental conditions no evidence of an anti-mitogenic action of BrdU was observ ed. We also compared the performance of the MTS assay and BrdU ELISA in mea suring substance P-induced CD4 + T cell proliferation. The results indicate d that the MTS assay may reflect change in cell activation leading to an ov erestimate of cell growth. In conclusion, our results indicate that the Brd U ELISA is the most sensitive of the three proliferation assays used for th e assessment of CD4 + T lymphocyte growth and is the preferred assay when s mall changes in cell growth are expected. (C) 1999 Elsevier Science B.V. Al l rights reserved.