Home › Microscopy & Imaging › Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay
Microscopy & Imaging JoVE (Open Access) Citable · DOI

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

DOI: 10.3791/53157-v
What you'll learn
  • ✓ Set up single-molecule FRET microscopy to measure TCR-pMHC binding kinetics
  • ✓ Prepare functionalized planar supported lipid bilayers with peptide-loaded MHC
  • ✓ Analyze immunological synapse dynamics using fluorescence recovery and photobleaching
  • ✓ Quantify binding constants from FRET efficiency measurements in living T cells
Protocol

This manuscript describes how to conduct (single molecule) Förster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

Difficulty
advanced
Total time
~3–5 days (protein production + cell preparation + microscopy acquisition + analysis)
Model organism
Jurkat T cells (human T-cell line)
Biosafety
BSL-1

Steps

1
Introduction to TCR-pMHC FRET binding assays

Understand the rationale for measuring single-molecule TCR-pMHC interactions within the immunological synapse using FRET-based microscopy on supported lipid bilayers.

▶ 00:56
2
Produce fluorescently labeled TCR and pMHC proteins

Express and purify recombinant TCR and peptide-loaded MHC molecules, labeling them with FRET donor and acceptor fluorophores.

▶ 03:00
3
Measure calcium flux in activated T cells

Monitor intracellular calcium mobilization in T cells upon TCR engagement to validate biological responsiveness of the system.

▶ 04:35
4
Perform FRET donor recovery after acceptor photobleaching

Measure FRET efficiency by selectively photobleaching acceptor fluorophores and tracking donor fluorescence recovery to quantify binding interactions.

▶ 06:27
5
Acquire and analyze single-molecule FRET trajectories

Image individual TCR-pMHC binding events in the immunological synapse and extract dwell times and FRET efficiencies from fluorescence time series.

▶ 11:07
6
Determine the FRET calibration constant C

Calculate the instrument-specific constant relating FRET efficiency to binding affinity using reference standards or theoretical models.

▶ 16:29
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