Real-Time Analysis of Resident T-cell Migration in Different Tumor Regions Using Confocal Microscopy

In This Article

Abstract

Source: Peranzoni, Elisa, et al. Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy. J. Vis. Exp. (2017).

This video describes the imaging of resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Protocol

1. Immunostaining of resident CD8 T cells and tumor cells

Dilute the antibodies (final concentration 10 µg/mL) and the nuclear dye (final concentration 1 µg/mL) in RPMI-1640 media without phenol red.
NOTE: Use fluorescently-coupled anti-CD8 (murine IgG, clone SK1) and anti-EpCAM (epithelial cell adhesion molecule) (murine IgG HEA-125) antibodies to label CD8 T lymphocytes and tumor epithelial cells, respectively. Use fluorescently-coupled anti-CD90/Thy1 (murine IgG, clone 5E10) antibody to label fibroblasts and activated endothelial cells. Use 4',6-diamidino-2-phenylindole (DAPI) to label the nucleus of ce....

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Results

Cell culture setup: RPMI medium, peristaltic pump, 35mm dish, microscope observation, solution flow.
Figure 1: Photographs of the perfusion system. A) The phenol red-free RPMI solution is enriched in 5% CO₂, 95% O₂. B) The solution is then perfused by a peristaltic pump. C) The RPMI solution enters the culture dish via the inlet. .......