The overall goal of this procedure is to surgically remove lymph nodes from an anesthetized mouse. This is accomplished by first localizing the lymph nodes. The lymph nodes are harvested, and then the incision is closed.
Finally, a single cell suspension can be obtained for experimental analysis. Ultimately, viable lymphocyte populations can be evaluated by flow cytometry. Visual demonstration of this technique is critical as lymph node localization and removal need to be viewed in order to be mastered.
After anesthetizing the mouse, apply eye ointment, inject buprenorphine and confirm sedation by toe pinch, then turn the mouse onto its side. First, localize the brachial lymph node and then shave the region to be dissected. After brushing away any loose hairs, apply a disinfectant to the region and then make a tiny incision of about five millimeters on the back of the four Legg.
Use two pairs of forceps to stretch the incision until the brachial lymph node, which can appear grayish or darker than the surrounding fat can be seen. Then pinch the fascia covering the lymph node with one pair of forceps. Place the second pair of forceps as far underneath the lymph node as possible and pull lightly on the membrane without breaking the surrounding tissue.
Then use the first pair of forceps to break the fascia and remove the lymph node. If the lymph node has been successfully harvested, a stain of blood will appear where the lymph node was previously located. Place the excised lymph node into an isotonic solution if it sinks, this will confirm the harvested tissue is a lymph node not fat.
Next, verify that no hairs are in the wound and close the incision by sticking the two insides of the skin together and by stapling them with one to two clips, depending on the size of the incision. Now localize a inguinal lymph node and shave the region to be dissected. After brushing away any loose hairs, apply a disinfectant to the region and then make a tiny incision of about five millimeters above the hind paw near the abdomen.
After opening the incision with two pairs of forceps as just shown for the brachial lymph node, identify the inguinal lymph node nestled within the surrounding fat tissue. Then remove the lymph node with the two pairs of forceps as just demonstrated. Again, confirm successful harvesting of lymph node by noting the stain of blood that appears where the lymph node was previously located, and then drop the inguinal lymph node into a separate container of isotonic solution.
After all the lymph nodes have been harvested, place the mouse into a cage and add wet food so the mouse can feed easily. After the surgery about six to eight hours later, check the mouse to ensure it is not experiencing pain. Carefully observe that posture, gait interaction with congen and appearance of the fur is similar to that of a normal healthy mouse.
Finally, dissociate the lymph node by pressing it against frosted slides in a Petri dish and then transfer it into a tube to obtain a single cell suspension. After staining the cells for the cell surface antigens of interest, analyze the cells by flow cytometry. Here, sample forward and side, scatterplots are shown for lymph nodes harvested from either anesthetized or sacrificed animals.
The profiles and percentages of cells in the live lymphocyte gates of the two scatterplots are the same. Demonstrating that lymph node surgery is an appropriate technique to harvest cells Once mastered the lymph node surgery can be done in five to 10 minutes per mouse if it is performed properly.
