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A dye-based lymphocyte proliferation assay that permits multiple immunological analyses: mRNA, cytogenetic,
Y Zhi-Jun1, N Sriranganathan, T Vaught
1Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University (Virginia Te.
Insights
Alamar Blue offers a non-radioactive method to assess lymphocyte proliferation, providing stable results and allowing further analysis of the same cells. This assay avoids cell lysis, unlike traditional methods, enabling comprehensive immunological studies.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Assessing lymphocyte proliferation is crucial in immunological studies.
- Traditional methods like the 3H-thymidine incorporation assay require cell lysis, limiting further analysis.
- A non-radioactive, non-lytic assay is needed to maximize information from cellular studies.
Purpose of the Study:
- To establish and validate Alamar Blue as a non-radioactive colorimetric assay for indirect determination of murine lymphocyte proliferation.
- To demonstrate that lymphocytes assessed with Alamar Blue can be re-utilized for subsequent immunological analyses.
- To confirm that Alamar Blue exposure does not detrimentally affect lymphocyte viability or interfere with various downstream assays.
Main Methods:
- Developed a colorimetric assay using Alamar Blue dye to measure lymphocyte proliferation.
- Compared Alamar Blue assay results with the standard 3H-thymidine incorporation assay.
- Washed Alamar Blue dye from cells after proliferation assessment and performed further immunological analyses, including apoptosis assays, flow cytometry, and mRNA expression analysis.
Main Results:
- Alamar Blue assay results for mitogen-induced lymphocyte proliferation were comparable to the 3H-thymidine incorporation assay.
- Color/fluorescence changes from Alamar Blue were stable for 3-4 weeks at 4°C in the dark.
- Short-term exposure to Alamar Blue did not harm lymphocytes, assessed by trypan blue exclusion and propidium iodide assays.
- Alamar Blue exposure did not interfere with apoptosis assays, chromosomal aberration analysis, cell surface antigen visualization, or cytokine mRNA expression.
Conclusions:
- Alamar Blue provides a reliable, non-radioactive method for assessing lymphocyte proliferation.
- This assay allows for subsequent immunological analysis of the same cells, maximizing experimental data.
- The Alamar Blue assay enhances immunological research by enabling correlation of proliferation with other cellular events in the same cell population.
Abstract:
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.
