1Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), 2Inserm, U1016, Paris, France
Salmon, H., Rivas-Caicedo, A., Asperti-Boursin, F., Lebugle, C., Bourdoncle, P., Donnadieu, E. Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes. J. Vis. Exp. (53), e3054, doi:10.3791/3054 (2011).
Naïve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.
1. Preparing slices from lymph nodes
2. Isolating T cells from mouse lymph nodes
3. Labeling of isolated T cells
Other fluorescent dyes than CMFDA might be used too, but keep in mind that these molecules are potentially toxic above certain concentrations.
4. Plating labeled T cells onto lymph node slices
5. Imaging T cells within lymph node slices
In this video-article, we image fluorescent T cells in lymph node slices with a widefield inverted microscope and a confocal upright microscope.
Imaging T cells with a wide-field inverted microscope
A typical time-lapse imaging experiment captures 5 optical planes spanning a total depth of 50 μm in the axial (z) dimension. Fluorescent T cells are usually imaged at intervals ranging 10 to 30 seconds during 10 to 20 min.
Imaging T cells with a confocal upright microscope
The confocal microscope used in this protocol is a Leica SP5 equipped with a 20x water immersion objective (Olympus, 20x/0.95 NA).
6. Representative results
By following this video-protocol you should expect to visualize a large number of fluorescent T cells accumulated in the T zone of the node, a phenomenon that normally occurs in this particular environment in vivo (Figure 1). In particular, T cells should be excluded from B cell zones, which are usually just beneath the capsule. A successful experiment will also result in T cells recruited into the tissue (Figure 2), displaying a highly motile behavior (Figure 3) consistent with published results obtained in intact lymph nodes 4. On average, the mean velocity of individual T cells within slices should be close to 10 μm/min (Figure 4).
Figure 1. T cells accumulate in a lymph node slice. Fluorescently-labeled T cells (CMFDA, green) were added to a lymph node slice 30 min before image acquisition (top picture). The image is the maximum projection of 5 images spanning 50 μm in the z direction. The bright field image is shown in bottom. Images were captured with a widefield microscope.
Figure 2. T cells are recruited into a lymph node slice. Fluorescently-labeled T cells (CMFDA, green) were added to a lymph node slice 30 min before image acquisition using a confocal microscope. Images were captured at the cut surface (top picture) and 40 μm below (bottom picture).
Figure 3. T cells are highly motile within a lymph node slice. Fluorescently-labeled T cells were imaged during 12 min using a widefield microscope. Trajectories of individual T cells are displayed as color-coded tracks to represent increasing displacements from blue (low motile cells) to red (high motile cells). Tracks were calculated using Imaris software. The white line follows the edge of the node, whereas the dashed oval delimits the putative B cell zone.
Figure 4. Within a lymph node slice, the T cell velocity is close to 10 μm/min. The speeds were calculated using Imaris software from tracks represented in Figure 3.
We have described a straightforward, quick and robust technique for generating lymph node slices, which are used to investigate the behavior of introduced T cells. In recent years, this method has been applied successfully using thymic and lymph node slices to identify the extracellular factors controlling T cell positioning and motility 1,5. It has also been used to measure Ca2+ responses in thymocytes during positive selection and in T cells upon antigen recognition 2,6. The overlay slice assay presents advantages and limitations that deserve to be discussed. Of note, this system permits access to the tissue, useful if one needs to manipulate slices pharmacologically in order to interfere with the molecular control of cell migration. Subsequent to imaging, the slices can be processed for immunohistochemistry to collect further information about the structures that have been imaged. Moreover, observation can be made with a traditional widefield fluorescence microscope. Although the resolution is not as good as with a confocal or a two-photon microscope and that phototoxicity is in principle more severe with one-photon than with two-photon microscopy, it has the advantages of simplicity, a lower cost, and larger choice of excitation wavelengths.
The imaging of T cells in an intact lymph node requires intravenous injection of labeled lymphocytes that subsequently home to lymphoid organs. As we have shown previously 1, the slice assay is perfectly compatible with adoptive transfer experiment. However, the length of time needed for T cells to home into lymph nodes can complicate the use of fluorescent dyes that have a tendency to leak out of the cells over time. This is the case of the Ca2+ dye fura-2. With the fast recruitment (< 30 min) of T cells into the tissue, the slice assay provides an opportunity to use such dyes. Finally, this method has the great advantage to analyze T cell functions in several human tissues kept alive.
This experimental system presents also limitations that need to be kept in mind. Damage associated with the slicing may affect T cell functioning, especially in the superficial region of the tissue near the cut surface. In order to assess cell death within the slice, we have used the fluorescent dye SYTOX green that penetrates cells with compromised plasma membrane. Our experiments reveal that about 20% of total nodal cells, mostly localized in the superficial region of the tissue, were fluorescently labeled with this nuclear dye. Another potential concern with the assay is the ability of the slices to retain important soluble factors including chemokines. Although, we have no indication that our data were affected by such problems since T cells display a good motility within the slice, we would like to stress the importance of imaging T cells in healthy regions located at several tens of microns from the cut surface. Whereas, this could be done with traditional microscopes (widefield or confocal) like in this protocol, it is likely that the lymph node slice preparation combined with two-photon imaging will increase the spatial resolution in depth and reduce the phototoxicity.
No conflicts of interest declared.
The authors would like to thank Dr. Alain Trautmann who encouraged us to perform lymph node slices. This work was supported in part by grants from the Ligue Nationale Contre le Cancer, the Fondation pour la Recherche Médicale en France and the Association pour la recherche sur le Cancer.
|Confocal microscope||Leica Microsystems||SP5|
|Fine Forceps||World Precision Instruments, Inc.||14142|
|30 mm Culture inserts||EMD Millipore||PICM0RG50|
|RPMI||Invitrogen||61870010||Complete RPMI-medium is made by adding 10 % heat-inactivated fetal calf serum and Penicillin/streptomycin|
|Hanks’ Balanced Salt Solution (HBSS)||Invitrogen||14170088|
|Low gelling temperature Agarose, type VII-A||Sigma-Aldrich||A0701|
|Butyl Cyanoacrylate Glue, Vetbond||3M||1469|
|Stainless steel washers, 4 mm of inner diameter||Any Supplier|
|Cell Tracker green CMFDA||Invitrogen||C7025|