Analysis of Immune Synapses between Human T Cells and SEB-Loaded Raji Cells using Imaging Flow Cytometry

Take flow cytometry tubes containing unloaded Raji cells and Staphylococcus aureus enterotoxin B or SEB-loaded Raji cells.

These SEB-loaded Raji cells function as surrogate antigen-presenting B cells containing the superantigen SEB complexed to class II MHC molecules.

Incubate with pan-leukocytes.

The T cell receptors, TCRs on T cells bind to the SEBs on Raji cells, which, along with co-stimulatory signals, establish a stable cell-cell junction, an immune synapse, leading to T cell activation.

This triggers intracellular signaling pathways, causing the actin cytoskeleton rearrangement, with the clustering of signaling molecules and surface receptors within the synapse.

Post-incubation, fix the cells to preserve their cellular structures. Permeabilize the cells to access intracellular targets.

Add fluorophore-labeled anti-CD3 antibodies, phalloidin, and DAPI, which bind to CD3 in the TCR complex, actin filaments, and DNA, respectively.

Using an imaging flow cytometer, visualize fluorescent signals from CD3 and actin to determine their enrichment at the synapse in the presence of SEB.