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An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers. The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.

T cell-mediated immunity plays an important role in controlling flavivirus infection, either after vaccination or after natural infection. The "quality" of a T cell needs to be assessed by function, and higher function is associated with more powerful immune protection. T cells that can simultaneously produce two or more cytokines or chemokines at the single-cell level are called polyfunctional T cells (TPFs), which mediate immune responses through a variety of molecular mechanisms to express degranulation markers (CD107a) and secrete interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, or macrophage inflammatory protein (MIP)-1α. There is increasing evidence that TPFs are closely related to the maintenance of long-term immune memory and protection and that their increased proportion is an important marker of protective immunity and is important in the effective control of viral infection and reactivation. This evaluation applies not only to specific immune responses but also to the assessment of cross-reactive immune responses. Here, taking the Japanese encephalitis virus (JEV) as an example, the detection method and flow cytometry color scheme of JEV-specific TPFs produced by peripheral blood mononuclear cells of children vaccinated against Japanese encephalitis were tested to provide a reference for similar studies.