Biology Reference
In-Depth Information
21.3.5 Preparation of Cell Surface Replicas .......................................... 401
21.3.5.1 Preparation of the Freeze-Fracture Unit............................ 401
21.3.5.2 Mounting of Samples on the Specimen Table ................... 402
21.3.5.3 Etching and Replication ................................................... 402
21.3.6 Cleaning and Mounting of Replicas ............................................ 403
21.3.7 Analysis of Metal Replicas by TEM............................................. 404
21.4 Analysis .........................................................................................................405
21.5 Considerations................................................................................................405
21.5.1 Safety Precautions.................................................................... 405
21.5.2 Starting Number of Cells........................................................... 405
21.5.3 Colloidal Gold Conjugates.......................................................... 406
21.5.4 Cell Surface Replication and Replication of Fracture Surfaces...... 406
21.5.5 Primary Antibody and Fixation Conditions................................... 406
21.5.6 Secondary Reagents ................................................................. 406
21.5.7 Freezing of Samples ................................................................. 407
21.5.8 Mounting of Samples in the Specimen Table .............................. 407
21.5.9 Replica Floating ....................................................................... 407
21.5.10 Replica Stability....................................................................... 407
21.5.11 Replica Quality......................................................................... 408
21.5.12 Low Labeling Efficiency and Implications for
Interpretation of Data ............................................................... 408
Acknowledgments ................................................................................................... 408
References ............................................................................................................. 408
Abstract
T cells show high sensitivity for antigen, even though their T-cell antigen receptor
(TCR) has a low affinity for its ligand, a major histocompatibility complex molecule
presenting a short pathogen-derived peptide. Over the past few years, it has become
clear that these paradoxical properties rely at least in part on the organization of cell
surface-expressed TCRs in TCR nanoclusters. We describe a protocol, comprising
immunogold labeling, cell surface replica generation, and electron microscopy
(EM) analysis that allows nanoscale resolution of the distribution of TCRs and other
cell surface molecules of cells grown in suspension. Unlike most of the light
microscopy-based single-molecule resolution techniques, this technique permits
visualization of these molecules on cell surfaces that do not adhere to an experimen-
tal support. Given the potential of adhesion-induced receptor redistributions, our
technique is a relevant complement to the substrate adherence-dependent techniques.
Furthermore, it does not rely on introduction of fluorescently labeled recombinant
molecules and therefore allows direct analysis of nonmanipulated primary cells.
