The overall goal of this fluorescent cell tracker based assay is to monitor and characterize specific cell populations of interest as they migrate from the skin to the draining lymph node after foot pad infection with Bacille Calmette-Guerin, or BCG. We have used this technique to identify the dendritic cell population that home to the draining lymph node after BCG infection of the skin. This technique allowed to study the cell migration by injecting the tumors during a defined 24-hour window.
Visual demonstration of this method is critical as the foot pad injection and lymph node isolation steps are difficult to learn from text. For microbacterium Bovus BCG injection, after confirming the appropriate level of sedation by toe pinch, use a syringe fitted with a 29-gauge 1/2 inch needle to inoculate the hind foot pad of an eight to 12 week old mouse with one times ten to the sixth colony-forming units of the bacteria in 30 microliters of PBS. Inject control foot pads with PBS only.
Then monitor the animals until they are fully recumbent and able to put weight on the injected foot pad. For CFSE injection, at the appropriate time point after BCG injection, dilute a vial of room-temperature, five millimolar CFSE tenfold in PBS, taking care to re-suspend the solution thoroughly. Then, load the syringe with the diluted CFSE and administer 20 microliters into the same hind foot pad that received the first injection.
24 hours after CFSE injection, carefully make a vertical incision in the hind thigh, and clear the muscle fat. Next, use scissors and tweezers to expose and excise the Popliteal Lymph Node, located deep within the fat pouch in the hollow of the knee. Transfer the lymph node to 0.5 milliliters of sterile PBS on ice, and homogenize the lymph node through a 70 micron cell strainer inside one well of a six well plate containing five milliliters of PBS with the back end of a three milliliter syringe.
Wash the strainer with another 5 milliliters of buffer, and transfer the cell solution into a 15 milliliter tube. Then, centrifuge the cells and resuspend the pellet in a sufficient volume of buffer. To label the cells for flow cytometry, I'll Liaquat one to ten times ten to the sixth cells into five milliliter round-bottom polystyrene tubes and wash the cells in fresh FACS buffer.
Re-suspend the pellets in 50 to 100 microliters of the appropriate antibody cocktail containing anti-mouse CD16 CD32 to block unspecific FC mediated interactions in FACS buffer on ice, protected from light. After 30 to 45 minutes, wash the cells in FACS buffer and re-suspend the pellets in 50 to 100 microliters of fresh FACS Buffer. The majority of CFSE labeling in the popliteal lymph node is observed in MHC-II high and CD11c positive low cells, consistent with skin dendritic cells that migrate to skin-draining lymph nodes.
Both the frequency and total number of these labeled cells appear in increased numbers in popliteal lymph nodes from BCG-infected mice, compared to PBS-injected controls, suggesting that BCG enhances the relocation of these cells to the draining lymph node. Skin dendritic cells continue to enter the draining lymph up to five days after infection, extending the migration of dendritic cells in the current model well beyond the timeline for the initial activation of antigen-specific CD4 positive T-cells in the popliteal lymph node. Further, migratory skin dendritic cells express elevated levels of co-stimulatory molecules, supporting the concept that these migratory dendritic cells are activated.
Importantly, both lymph node resistant mono-cytoid and plasma-cytoid dendritic cells, which enter inflamed lymph nodes through high endothelial venules, and not afferent lymphatics, are negative for CFSE, further verifying that CFSE labeling occurs mainly in the foot pad. Moreover, additional flow cytometric analysis reveals that a sub-population of EpCAM low CD11b high cells is the main migratory skin dendritic cell subset that drains to the popliteal lymph node in response to BCG infection. Once mastered, this technique can be performed in five minutes if it is done properly.
While attempting this process, it's important to be careful with the injection site and the injection volume;much care should be taken not to damage the draining lymph node during dissection. Following the procedure, other methods like flow cytometry or confocal microscopy can be performed for further analysis of subset-specific cell micro expression. This technique paved the way for researchers to track cells from the skin to the draining lymph node, within a 24 hour period in response to infection or vaccination.
After watching this video, you should have a good understanding of how to perform foot pad injections and to locate and isolate the draining lymph nodes for tracking and characterization of migratory dendritic cell populations of interest.
