Evaluation of Controlled T Cell Activation with a Photoactivatable Peptide MHC

Abstract

Source: Sanchez, E., et al. Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist. J. Vis. Exp. (134), (2018)

This video demonstrates a method of decaging a photoactivatable peptide-major histocompatibility complex using UV irradiation. The decaging process exposes the native peptide sequence, enabling precise T cell activation.

Protocol

1. Preparation of Stimulatory Glass Surfaces

1. Coat eight-well chambered coverglass with biotinylated poly-L-lysine (Bio-PLL) diluted 1:500 in distilled, deionized water (ddH₂O). Incubate for 30 minutes at room temperature (RT).
2. Wash with H₂O.
3. Dry for 2 h at RT.
4. Block Bio-PLL coated surfaces with blocking buffer (HEPES-buffered saline [10 mM HEPES pH 7.4, 150 mM NaCl], with 2% BSA) for 30 min at RT.
   1. Dissolve 5 mg of poly-L-lysine hydrobromide in 1 mL of 10 mM NaPO4, pH 8.5.
   2. Add 125 μmol (0.55 mg) of NHS-biotin (from a 100 mg/mL stock in DMSO) and check the pH of ....

Results

Figure 1: Microscope configuration for photoactivation and TIRF imaging.

488 nm and 561 nm lasers are used for TIRF and epifluorescence imaging of green and red probes, respectively. UV light for photoactivation is taken from a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nunc Lab-Tek Chambered Coverglass	Thermofischer Scientific	155361
Poly-L-lysine hydrobromide	Sigma-Aldrich	P2636	Will need to make Biotinylated Poly-L-Lysine
EZ-Link NHS-Biotin	Thermofischer Scientific	20217	Will need to make Biotinylated Poly-L-Lysine
Streptavidin	Thermofischer Scientific	434301
BirA-500: BirA biotin-protein ligase standard reaction kit	Avidity	BirA500	Will be used to biotinylate proteins
Biotinylated Hb I-EK			For protein folding, see reference 6. For biotinylation, use BirA kit
Biotinylated NPE-MCC I-EK	Anaspec		Custom NPE-MCC (HANERADLIAYL-K(Nvoc)-QATKOH) can be purchased from Anaspec
Biotinylated αH2-Kk antibody	BD Biosciences	553591
Biotinylated NPE-OVA H2-Kb	Anaspec		Custom NPE-OVA (H-SIINFE- K(Nvoc)-L-OH) can be purchased from Anaspec
Biotinylated KAVY H2-Db	Anaspec		Custom synthesized protein (KAVYDFATL) can be purchased from Anaspec
Biotinylated ICAM1			For protein folding, see reference in protocol. For biotinylation, use BirA kit
Hand held UV lamp	UVP	UVGL-25	Lamp is held < 1 cm from the sample. 30 s of 365 light is sufficient for detectable decaging, 20 min for quantitative decaging.
Olympus IX-81 OMAC TIRF system.	Olympus		Additional information about the imaging system can be found in Figure 6
Mosaic digital diaphragm	Andor
Slidebook software	Intelligent Imaging Innovations