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

Dilute the required fluorescently labeled antibodies and the fluorescence nuclear dye, DAPI, to the required final concentrations in phenol red-free RPMI medium. Next, remove the culture plate from the incubator and using a fine tip, aspirate out the liquid inside the stainless steel washers.

Now, without touching the slice, add 40 microliters of the diluted antibodies onto each tumor slice. To allow for staining, incubate the plate at 37 degrees Celsius for 15 minutes. After the staining step is complete, using fine forceps, remove the washers. Then, gently take out the slices and dip them in phenol red-free RPMI1640 medium for 10 seconds. Place the slices back onto the cell culture inserts and add a drop of phenol red-free RPMI1640 medium over each slice. Incubate the plate at 37 degrees Celsius for 10 minutes and then proceed to imaging.

Several hours prior to starting this experiment, set the temperature of the microscope heat chamber to 37 degrees Celsius, then, prepare the microscope set up to constantly perfuse tumor slices with oxygenated phenol red-free RPMI medium, and to aspirate the solution to a waste collection flask using a peristaltic pump.

With the help of fine forceps, lift the stained slice out of the six-well plate, and transfer it to a 35-millimeter plastic dish filled with phenol red-free RPMI medium, then, place a slice anchor over the slice to support it. Next, place this Petri dish on the imaging stage of the microscope.

After connecting the inlet and outlet tips of the perfusion system, turn on the peristaltic pump to allow the oxygenated medium to go through the perfusion tubes. Lower the water immersion objective to the slice, and focus on the top of the slice with bright-field light or with the appropriate wavelength.

Then, using appropriate filters, visualize and select a region of interest that contains all the fluorescently labeled cells of interest namely the CD8 T cells, the tumor islets, and the CD90-positive stromal cells. Set up an imaging session using appropriate laser intensity and exposure times to suit the brightness of the fluorescent signals observed. Then, set up the Z-stack thickness to image within the tumor slice.

Next, select the Z-stack image time interval and the total recording time to capture images at specific time intervals. After capturing and exporting the fluorescent images, analyze CD8 T cell migration as described in the text protocol.