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Abstract: Objective: In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4⁺ CD25⁺ regulatory T (Treg) cells, in order to inform further studies in Treg cell function. Methods: We separately used magnetic cell sorting and flow cytometry sorting to identify CD4⁺ CD25⁺ Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4⁺ CD25⁺ Treg cells, trypan blue staining to detect cell viability, and propidium iodide (PI) staining to assess the cell viability. We detected the immune inhibition of CD4⁺ CD25⁺ Treg cells in the in vitro proliferation experiments. Results: The results showed that compared to flow cytometry sorting, magnetic cell sorting took more time and effort, but fewer live cells were obtained than with flow cytometry sorting. The CD4⁺ CD25⁺ Treg cells, however, obtained with both methods have similar immunosuppressive capacities. Conclusion: The result suggests that both methods can be used in isolating CD4⁺ CD25⁺ Treg cells, and one can select the best method according to specific needs and availability of the methodologies.

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