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.
1. Immunostaining of resident CD8 T cells and tumor cells
2. Imaging and analyzing resident CD8 T cell migration within human lung tumor slices
NOTE: The confocal microscope used in this protocol is an upright spinning disk equipped with a 25x water immersion objective (25x/0.95 N.A.).
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 determine solution height.
The authors have nothing to disclose.
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 |