A CFSE-Based Assay to Monitor Skin Dendritic Cell Migration to Draining Lymph Nodes in Infected Mice

For Mycobacterium bovis BCG injection, after confirming the appropriate level of sedation by toe pinch, use a syringe fitted with a 29-gauge half-inch needle to inoculate the hind footpad of an 8- to 12-week-old mouse with 1 x 10⁶ colony-forming units of the bacteria and 30 microliters of PBS. Inject control footpads with PBS only. Then, monitor the animals until they are fully recumbent and able to put weight on the injected footpad.

For CFSE injection, at the appropriate time point after BCG injection, dilute a vial of room temperature 5-millimolar CFSE tenfold in PBS, taking care to resuspend the solution thoroughly. Then, load the syringe with the diluted CFSE, and administer 20 microliters into the same hindfoot 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 5 milliliters of PBS with the backend of a 3-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, aliquot 1 to 10 x 10⁶ cells into 5-milliliter round round-bottom polystyrene tubes, and wash the cells in fresh FACS buffer. Resuspend the pellets in 50 to 100 microliters of the appropriate antibody cocktail containing anti-mouse CD16/CD32 to block unspecific Fc-mediated interactions and FACS buffer on ice, protected from light. After 30 to 45 minutes, wash the cells in FACS buffer, and resuspend the pellets in 50 to 100 microliters of fresh FACS buffer.