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