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

Published: July 31, 2023

Abstract

Source: Bello-Gamboa, A. et al., Imaging the Human Immunological Synapse. J. Vis. Exp. (2019)

This video demonstrates the in vitro study of immunological synapse formation between an antigen-presenting cell and helper T lymphocytes using a fluorescence microscope. T cell receptors of helper T lymphocytes interact with an antigen-MHC-II complex in antigen-presenting cells and form immunological synapses, followed by secretory vesicle movements toward the synapse, which is visualized by fluorescent microscopy.

Protocol

1. Preparation of Slides to Adhere Raji Cells Add 150 µL of fibronectin (100 µg/mL) per well to an 8 microwell chamber slide (plastic-bottom chamber slide) and incubate it for 30 min to 1 h at 37 °C. This adhesion substrate will allow the binding of Raji cells to the well bottom (Step 2), the formation of living conjugates with Jurkat cells (Step 4) cells, and time-lapse microscopy capture (step 6), and is also compatible afterward with optional paraformaldehyde (PFA) fixation….

Representative Results

Figure 1 represents synaptic Jurkat-Raji conjugates that were obtained following the protocol (step 5.2). The image represents the first frame from a representative, time-lapse experiment. 2 x 105 Raji cells and 2 x 105 Jurkat cells were added to a 1 cm2 well. The upper panel shows transmittance channel, the middle panel consists of CMAC (blue) chan…

Divulgazioni

The authors have nothing to disclose.

Materials

Camera Nikon DS-QI1MC Nikon MQA11550 Cooled Camera Head
CMAC ThermoFisher Scientific C2110 Cell tracker blue
JURKAT cells ATCC ATCC TIB-152 Effector T lymphocytes
μ-Slide 8 well ibiTreat, μ-Slide 8 well Glass-Bottom IBIDI Cat.No: 80826, 80827 Cell culture and cell imaging supports
Microscope NIKON Eclipse Ti-E Nikon NIKON Eclipse Ti-E Wide-field fluorescence, fully-motorized microscope equipped with Perfect Focus System (PFS) option
Microscope Stage Incubator with 3-channel manual gas mixer and gas bubbler/ humidity module OKOLAB H201-NIKON-TI-S-ER Cell culture atmosphere
Raji Cells ATCC ATCC CCL-86 APC
RPMI medium GIBCO ThermoFisher Scientific 21875034 Culture medium
Streptococcus Enterotoxin E (SEE) Toxin Technology, Inc EP404 Bacterial Toxin
Software Image J NIH Image J Image software
Software Nikon NIS-AR Nikon NIS-Elements AR Image capture and analysis software

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Citazione di questo articolo
Immunological Synapse Formation Between Helper T Cells and Antigen-Presenting Cells. J. Vis. Exp. (Pending Publication), e21558, doi: (2023).

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