The overall goal of this procedure is to image fluorescent T cells in lymph node slices by widefield and confocal microscopy. This is accomplished first by removing a mouse lymph node and embedding it in low melting point aero. The node is then cut into 320 micron thick sections with a vibrato.
Next fluorescently dyed T cells are plated on top of a lymph node slice. Finally, the T-cell that have infiltrated the slice are visualized by widefield and confocal microscopy. Ultimately still and videographic images can be obtained that show how T cells are recruited into the lymph node and what kind of motile behavior they exhibit within the lymph tissue.Okay.
The main advantage of these techniques of a existing metal, like two photomicroscopy and intact organs, is that here the T cells can be visualized in a three-dimensional system with a conventional white field microscope. And moreover, the the slight preparations, permits and access to the tissue issue Begin by preparing a 4%low melting point. Arose solution by diluting arose in PBS and microwaving the resulting admixture after the arose has completely dissolved.
Maintain the solution at 37 degrees Celsius until use. Next, fill a plastic dish with RPMI. Complete culture media and place stainless steel washers with inner diameters of four millimeters into the dish.
Fill another dish with ice cold PBS then add approximately 1.1 milliliter of RPMI. Complete culture media per well in a six well tissue culture plate. Insert an organotypic filter into each well of the six well plate and place the plate of four degrees Celsius.
Carefully remove the peripheral lymph nodes from their surrounding tissues in a euthanized mouse. Dissect the lymph nodes carefully as they are very soft. Very important to remove all the fat from the tissue because it's very toxic for the cells.
Place the lymph nodes in the plastic dish containing the ice coal PBS. Now pour the 4%aro gel into a 35 millimeter plastic dish and delicately transfer the nodes into the gel. Then place the aro on ice for five minutes to harden after the aro has hardened, remove the block from the dish and trim the agar, leaving approximately three to five millimeters of gel around each lymph node.
Then use non-toxic glue to attach the embedded nodes onto the specimen disc of a viome. Install the specimen disc in the viome removable tray and fill the tray with ice cold PBS. Set the viome speed on a slow range and the vibration frequency on a medium range section the agar embedded tissue at 320 micron thickness.
Discard the first slice as it contains only superficial tissue, and then use fine forceps to carefully transfer the lymph node slices as they are being cut into the organotypic culture inserts. Place stainless steel washers on each individual slice, making sure the washers are on the agro, is surrounding not covering the lymph node tissue. Then incubate the culture plate with the lymph node slices at 37 degrees Celsius in a 5%carbon dioxide humidified incubator.
While preparing the T cells for this step, use overnight culture IL seven supplemented T-cell that were isolated by CD four positive bead selection using MIL 10 max purification as demonstrated in the previously published JoVE article by Matthew and Callaghan. Wash the TCELL in HBSS for five minutes at 300 times gravity at 20 degrees Celsius. Re suspend them in fresh HBSS at 10 times 10 to the six cells per milliliter.
Then add one micromolar cell tracker green C-M-F-D-A to a tube of HBSS and combine this solution with the cell solution to a final dye concentration of 0.5. Micromolar incubate the cell suspension for five minutes at 37 degrees Celsius. Then after washing the cells twice with complete RPMI medium for five minutes at 300 times G and 20 degrees Celsius, Reeses bend the labeled T-cells as a final concentration of 10 times 10 to the six cells per milliliter.
In fresh RPMI, complete culture medium, retrieve the lymph node slices from the incubator. Remove the excess medium contained in the center of the washer with a pipette without touching the tissue. With the tip of the pipette, gently dispensed 10 to 20 microliters of labeled T-cell into the center of the washer.
Be sure that the drop of cell suspension stays in place. Incubate the slices and T-cells for at least 30 minutes to allow the lymphocytes to migrate into the tissue. To begin imaging, set the temperature control chamber on the microscope to 37 degrees Celsius.
Fill up a fusion system for enabling continuous perfusion of the lymph node slice with oxygenated phenol red free RPMI medium fill and warm a small plastic dish with oxygenated RPMI at this time as well. In the system shown here, 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 retrieve the T-cell infiltrated lymph node slices from the incubator with fine forceps.Delicately. Remove a lymph node slice from the six well plate. Dip the slice into the warmed oxygenated RPMI medium for a few seconds to wash away any fluorescent cells that have not infiltrated the tissue.
Then lay the freshly washed slice upside down on the imaging 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.
Then secure the slice by placing a stainless steel washer on top of it to capture a large field of view. Collect images using a 10 times dry objective lens. Transfer the freshly washed lymph node slice face up in a plastic dish.
Place a washer on the preparation to immobilize it. The slices are now ready to be imaged. Install the preparation on the stage of the upright confocal microscope and focus with the 20 times objective.
Then start an imaging session and start the acquisition.C-M-F-D-A. Labeled T cells in green were added to a lymph node slice 30 minutes before image acquisition as seen here. The image is the maximum projection of five images spanning 50 microns in the Z direction captured with a widefield microscope here.
The same figure can be seen as a bright field image in this video. Green fluorescent T cells that have infiltrated a lymph node slice where image with a widefield microscope for nine minutes. The animation represents a 50 micron Z projection.
Note the active migration of T cells within the slice. The trajectories of individual T cells from the same microscope field are displayed here as color-coded trails to represent their increasing displacement from blue low motile cells to red high motile cells. The trails were calculated using a Mars.
The white line indicates the edge of the node, whereas the dashed ovals delineate the putative B cell zones. Here T cells added to a lymph node slice were image for 10 minutes with a confocal microscope. The animation represents a 50 micron Z projection.
Yeah, this technique can be adapted to other types of tissue. And what is interesting is that the slice as a is the only way to to explore mechanisms underlying T-cell migration and localization in human tumors. For example.
