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

Published: March 29, 2024

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 (muri…

Representative Results

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. At the outlet, the solution is aspirated by the pump from the chamber into a waste container. Note that the aspiration tip is set at a level to de…

開示

The authors have nothing to disclose.

Materials

Upright confocal microscope Leica The use of a spinning disk confocal is recommended
Imaris software Bitplane This sofware is used for visualization of images and tracking of T cell migration
Fine Forceps World Precision Instruments 14142
30 mm Culture inserts Millipore PICM0RG50
Roswell Park Memorial Institute (RPMI) medium, GlutaMAX Supplement ThermoFisher 61870010 Complete RPMI-medium is made by adding 10 % heat-inactivated fetal calf serum and Penicillin/ streptomycin
Phosphate-buffered saline (PBS) ThermoFisher 20012019
Hanks' Balanced Salt Solution (HBSS) Invitrogen 14170088
Low gelling temperature Agarose, type VII-A Sigma-Aldrich A0701
Non-toxic butyl Cyanoacrylate Glue, Vetbond 3M 1469
Slice anchors, 19.7 diameter, 2 mm spacing threads Warner Instruments 64-1415
Stainless steel washers, 5 mm of inner diameter Amazon B004K1FDGQ
BV605-conjugated anti-CD8 (clone SK1) BD Biosciences 565289
FITC-conjugated anti-EpCAM (clone HEA-125) Miltenyi Biotec 130-098-113
BV510-conjugated anti-CD90 (clone 5E10) Biolegend 328125
4',6-diamidino-2-phenylindole (DAPI ThermoFisher D1306

タグ

Play Video

記事を引用
Real-Time Analysis of Resident T-cell Migration in Different Tumor Regions Using Confocal Microscopy. J. Vis. Exp. (Pending Publication), e22069, doi: (2024).

View Video