Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

1. Preparing slices from lymph nodes

1. Prepare a 4% low-melting point agarose solution for embedding. Dilute agarose in PBS and microwave this solution. When the agarose is completely dissolved, maintain the solution at 37°C until ready for use.
2. Prepare a 6-well tissue culture plate. Add 1.1 ml RPMI complete culture medium per well, and insert an organotypic filter in each well. Place the 6-well plate at 4°C until ready to transfer slices.
3. Place stainless steel washers, with inner diameters of 4 mm, into a plastic dish filled with RPMI complete medium. Washers will be further used to concentrate the cells on the vibratome cut slice.
4. Sacrifice the mouse in accordance with local animal welfare regulations. Carefully remove the peripheral lymph nodes from the surrounding tissue and place them in a plastic dish containing ice-cold PBS. Care must be taken not to damage the lymph node structure, as these organs are very soft.
5. Pour the 4%-agarose gel into a 35-mm plastic dish and delicately transfer the nodes into the gel. Leave the agarose gel on ice for 5 min to harden.
6. Remove the block from the dish and trim the agar in order to leave 3-5 mm of gel around each lymph node.
7. Attach the embedded nodes on the specimen disk of the vibratome with non-toxic tissue glue. Install the specimen disk in the tray filled with ice-cold PBS.
8. Section the agar-embedded tissue at 320 μm thickness with the vibratome speed set on a slow range (0.3 mm/s) and the vibration frequency set on a medium range (1.5 mm).
9. Carefully transfer lymph node slices as they are being cut onto the organotypic culture inserts using fine forceps. Place 3 slices on each insert. Use great care as slices can be easily damaged. A typical peripheral lymph node will yield 5 sections when cut at 320 μm. Discard the first and last slices, as they only contain superficial tissue.
10. Place stainless steel washers on each individual slice. Make sure washers are well positioned on the agarose surrounding the tissue.
11. Incubate the culture plate at 37°C in a 5% CO₂ humidified incubator until ready to plate T cells.

2. Isolating T cells from mouse lymph nodes

1. Isolate T cells according to the previously published JoVE article ³.
2. Use T cells immediately and proceed to the next section. Otherwise, lymphocytes might be cultured overnight in complete RPMI-medium supplemented with 10 ng/ml IL-7.

3. Labeling of isolated T cells

1. Wash cells in HBSS. Resuspend them in the same buffer at 10 x 10⁶ per ml.
2. Add the same volume of HBSS containing 1 μM Cell Tracker green CMFDA giving a final concentration of 0.5 μM.
3. Incubate the cell suspension at 37°C during 5 min. Wash cells twice with complete RPMI-medium.
4. Resuspend cells at a final concentration of 10 x 10⁶ per ml in complete RPMI-medium. These labeled cells will be plated onto the slices.

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

1. Remove excess medium contained in the inner part of the washer with a pipette. Do not allow the tissue to become dry; work quickly during this step.
2. Gently dispense 10 to 20 μl labeled T cells that corresponds to 1 to 2 x 10⁵ cells in the inner part of the washer. Care must be taken not to touch the tissue with the tip of the pipette. Be sure that the drop of cell suspension stays in place.
3. Place the slices with cells in incubator at 37°C and 5% CO₂ for at least 30 min to allow lymphocytes to migrate into the tissue.

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.

1. Set the temperature at 37°C. Our microscopes are equipped with temperature-controlled chambers.
2. Install a perfusion system enabling continuous perfusion of the lymph node slice with oxygenated (5% CO₂, 95 % O₂) phenol red-free RPMI medium. In our system, perfused media enters into the imaging chamber from one side by gravity. The medium is aspirated with a pump connected to a waste collection flask. Set the media flow rate to 1 ml/min.
3. With fine forceps, delicately take out the slice from the 6-well plate and dip the preparation in warmed oxygenated RPMI medium for a few seconds to get rid of fluorescent cells not infiltrated into the tissue.

Imaging T cells with a wide-field inverted microscope

4. Place the slice upside down on a custom-made imaging chamber which consists of nylon threads glued onto the inner part of a glass bottom dish. In this chamber, the nylon threads elevate the slice from the glass and enable the oxygenated solution to diffuse into the tissue. This is required as T cell migration in lymph nodes is strongly dependent on oxygen. Secure the slice by adding onto it a stainless steel washer. In these conditions, the slice lies a few microns above the bottom of the dish, which facilitates renewal of the perfusion solution.
5. To capture a large field of view (800 x 800 μm, approximately one third of the node’s slice surface), collect images using a 10x dry objective lens. We found that the 10x Nikon S fluor 0.5 NA was most suitable for our analyses.

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. Secure the slice to the imaging stage of the microscope. Note: We usually place a washer on the preparation to immobilize it.
7. Set an imaging session by collecting a series of images along the z axis. To image T cells in an intact tissue structure, the start position is usually set at 5 to 10 μm below the first labeled T cell.

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.