Abstract
In adaptive immune system, formation of immunological synapse between T cells and antigen presenting cells (dendritic cells, B cells, and macrophages) or target cells (tumor cells and viral-infected cells) is critical for the execution of T cell immune responses via cytokine secretion or direct killing activity. Here, we describe the practical methods that directly measure the number of conjugates as a result of immunological synapse formation between T cells and superantigen-loaded B cells or between cytotoxic T cells and antigen-loaded target cells by dual-color flow cytometry.
Keywords: T cells, Antigen-presenting cells, Immunological synapse, Adhesion, Conjugation
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. SEE-induced Jurkat T cell-Raji B cell conjugates formation. Conjugates formation using Jurkat T cells and Raji B cells with or without SEE were performed as described in Procedure section A and then analyzed by flow cytometry. Dot plot showing FSC and SSC is gated to exclude cell debris and select cells for dual-color analysis (Left). Cells inside the gate were analyzed by CMFDA (green; T cell) and CMRA (red; B cell) fluorescence signals (middle dot plots). Quadrants are established using negative control and single-color controls. CMFDA+ CMRA- cells (lower right quadrant) indicate T cell only, CMFDA- CMRA+ cells (upper left quadrant) indicate B cell only, and CMFDA+ CMRA+ cells (upper right quadrant) indicate T cell-B cell conjugates. After loading of SEE by Raji B cells (+SEE), T cell-B cell conjugates were increased in compared with SEE-unloaded B cells (-SEE). Numbers in dot plots indicate the percentage of each quadrant. Confocal fluorescence image and differential interference contrast (DIC) image show T cell only (green box), B cell only (red box), and T cell-B cell conjugate (yellow box) after conjugation assay with SEE (Right).
Recipes
Acknowledgments
This work was supported by the Basic Science Program (2015R1A2A1A15052658), and the Creative Research Initiative Program (2015R1A3A2066253) through the National Research Foundation (NRF) grants funded by the Ministry of Science, ICT & Future Planning (MSIP), Korea, and Bioimaging Research Center at GIST.
References
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I don`t understand then how to do you quench the conjugation. As you know the acquisition and machine adjustment takes some time and in that particular duration other samples will have different stimulation time...Can you please share the gating strategy for the assay ? I am unable to understand your protocol as I am trying to use the same strategy but unable to get the same results as yours...