Immunological Synapse Formation Between Helper T Cells and Antigen-Presenting Cells

Retrieve the chamber slides containing the cell tracker blue-labeled Staphylococcus Enterotoxin E-pulsed adhered Raji cells from the incubator. Carefully aspirate the culture medium from each well, one by one, with a pipette placed in a corner of the well.

Immediately replace the medium with 200 microliters of the resuspended Jurkat cells in cell culture medium. If a time-lapse is being performed, quickly proceed to the microscope.

Locate the chamber slide on the microscope stage incubator and select some appropriate fields.

Prepare the microscope in incubation chamber prior to imaging. After the Jurkat cells have been added to each well containing the Raji cells, quickly locate the microwell chamber slide on the preheated microscope stage incubator, and select some XY positions.

If only an end-point experiment is planned, incubate the chamber slide at 37 degrees Celsius with 5% carbon dioxide for 1 to 2 hours. After the culture period, check for conjugate formation and subsequently fix the conjugates, as outlined in the text protocol.