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

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…

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