A dye-based lymphocyte proliferation assay that permits multiple immunological analyses: mRNA, cytogenetic, apoptosis, and immunophenotyping studies.

Alamar Blue in the microenvironment of activated cells, undergoes color change and also becomes fluorescent. By using the Alamar Blue dye, we have reported a non-radioactive colorimetric assay to indirectly determine proliferation of murine lymphocytes. We further show that the pattern of mitogen-induced proliferation assessed fluorometrically was comparable to the 3H-thymidine incorporation assay (3H-Tdr assay). Of practical importance is that the color/fluorescence changes were stable at 4 degrees C in the dark for 3-4 weeks. In immunological studies, it is important to further analyze lymphocytes that have undergone activation and/or proliferation. This is not possible with the standard 3H-Tdr assay, which requires lysis of cells. In contrast, the Alamar Blue-based non-radioactive assay does not require cell lysis. We therefore tested the hypothesis that further analysis of lymphocytes is possible, after assessing the proliferation using Alamar Blue. Following assessment of proliferation in a 72-h culture, the Alamar Blue dye was washed-off and cells were re-utilized to perform additional immunological analysis. Short-term exposure of lymphocytes to Alamar Blue was not detrimental to lymphocytes, as assessed by trypan blue exclusion and the propidium iodide (PI) assays. Exposure of dexamethasone-treated cells to Alamar Blue did not interfere with the performance of apoptosis assays, such as flow cytometric analysis of PI-stained cells and microscopic examination of ethidium bromide/acridine orange-stained cells. In addition, prior exposure of lymphocytes to Alamar Blue did not affect the analysis of chromosomal aberrations or the visualization of cell surface antigens by flow cytometry. Further, the expression of cytokine mRNA in lymphocytes previously exposed to Alamar Blue was similar to unexposed cells. Together, a notable advantage of this assay is that it now enables the investigator to maximize information by following or correlating proliferation with other immunologic events in the same cells.