Evaluation of Controlled T Cell Activation with a Photoactivatable Peptide MHC

Published: January 31, 2024

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 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). Wash with H2O. Dry for 2 h at RT. 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. Dissolve 5 mg of poly-L-lysine h…

Representative 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 mercury (Hg) lamp attached to a digital diaphragm system capable of illuminating small, user-defined regions. Light from the Hg lamp/diaphragm …

Divulgations

The authors have nothing to disclose.

Materials

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

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Citer Cet Article
Evaluation of Controlled T Cell Activation with a Photoactivatable Peptide MHC. J. Vis. Exp. (Pending Publication), e21880, doi: (2024).

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