Encyclopedia of Experiments: Immunology
Se requiere una suscripción a JoVE para ver este contenido. Inicie sesión o comience su prueba gratuita.
Transcript
Begin by adding biotinylated poly-L-lysine or bio-PLL diluted 1 to 500 in distilled deionized water to 8 8-well chambered cover glass. Incubate for 30 minutes at room temperature. Following the incubation, wash with distilled deionized water, and then dry for two hours at room temperature.
Block bio-PLL-coated surfaces with blocking buffer consisting of HEPES-buffered saline with 2% BSA for 30 minutes at room temperature. Following the incubation, remove the blocking buffer from the bio-PLL-coated surfaces without allowing the wells to dry. Then, add streptavidin, and incubate at 4 degrees Celsius.
After an hour, wash the coated surfaces in HBS. Fill and invert the chamber slide wells 4 to 5 times, removing HBS from the wells. Do not allow the wells to dry.
Add the specific biotinylated photoactivatable peptide major histocompatibility complex ligands and adhesion molecules for either CD4-positive T cells or CD8-positive cells to the bio-PLL-coated surfaces. Protect from light, and incubate for 1 hour at room temperature. After the incubation, wash as before, and leave in HBS until ready to seed. Finally, add 200,000 CD4-positive or CD8-positive T cells, expressing the appropriate TCR into the correct wells, and allow the cells to adhere at 37 degrees Celsius for 15 minutes.
Once the cells have attached to and spread on the surface, they are ready for photoactivation and imaging. Use an inverted total internal reflection fluorescence microscope outfitted with a UV-compatible 150x objective lens for image acquisition. Monitor probes in both the green and red channels using 488-nanometer and 561-nanometer excitation lasers respectively.
After mounting the chamber slide containing the T cells, adjust the microscope settings to obtain TIRF or epifluorescence illumination as necessary. In live mode, select a field of cells that are expressing the fluorescent probes of interest. Establish micron-scale regions for photoactivation beneath individual cells using software control. Then, start the acquisition.
Typically, 80 time points are acquired with an interval of 5 seconds between each. This leaves time for sequential 488-nanometer and 563-nanometer exposures in the case of dual-color experiments. Then, after acquiring 10 time points, photo-activate the selected regions by opening the digital diaphragm shutter for 1 to 1.5 seconds. After the time-lapse is complete, select a new field of cells, and repeat the process.
