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