Mouse Lymph Node Single Cell Suspension Preparation Process and Precautions

Preparation process of mouse lymph node single cell suspension

1)    Kill mouse by cervical dislocation, soak the body in 75% alcohol for 5 minutes and place it on a sterile operating table with the abdomen facing up.

2)    Use scissors to cut the skin from the sternum along the midline to the lower jaw, and then cut the skin from the lower jaw to the base of the left and right ears. Hold the skin with tweezers and lift it to left and right direction, fix the skin with a needle. After the above procedure you can see a pair of large submandibular glands above the sternum. There are yellow anterior cervical lymph nodes attached to the upper borders of the left and right submandibular glands. Cut off the sternocleidomastoid muscle and muscle belly, and lift up their two severed ends. It can be seen that there is a small deep cervical lymph node on the left and right in the deep dorsal part of the left submandibular gland. Remove lymph nodes carefully with forceps and small ophthalmic scissors.

3)    Remove the lymph nodes and immerse them in clean PBS solution.

4)    Aspirate the culture medium with a sterile 2.5 mL syringe, hold the lymph node with tweezers in the left hand, and hold the syringe in the right hand, carefully insert it into the lymph node and pipette until the lymph node cells are completely cleaned, and observe that only white connective tissue and adipose tissue remain.

5)    Filter the thymocytes by pipetting through a 200-mesh sieve, collect them in a 15 mL centrifuge tube, then centrifuge at 300 g for 5 min and discard the supernatant.

6)    Resuspend thymocytes in cell staining buffer, count the cells and adjust the cell concentration to 1×10⁷/mL.

FSC/SSC diagram of mouse lymph node cells

Precautions:

1.  Cervical lymph nodes

The superficial cervical lymph nodes are located on the superficial and lateral sides of the left and right submandibular glands, and are often surrounded by adipose tissue and are oval in shape. The deep cervical lymph nodes are located deep on the ventral side of the left and right sternomastoid and cleidomastoid muscles, and on the lateral side of the muscle group below the hyoid bone, that is, deep on the dorsum of the submandibular abdomen. During excision, the femoral artery can be punctured first for acute experiments, and the carotid artery can be cut off for bloodletting, so as to avoid bleeding during surgery and it is difficult to observe the location of the lymph nodes.

2.  Axillary lymph nodes

The axillary lymph nodes are located in the left and right armpit adipose tissue, pear-shaped. Cut the chest muscle along the sternum, lift it to the upper side, peel off the fat tissue in the armpit, and you can find this pair of larger lymph nodes.

3.  Brachial lymph node

The brachial lymph node are located in the superficial subcutaneous connective tissue, close to the left and right biceps muscle belly, oval shape, and can be found from the axilla to the outside along the subscapularis muscle.

4.  Intrathoracic lymph nodes

The intrathoracic lymph nodes are nearly round, and their number and location distribution vary greatly. Lymph nodes are generally observed in the following locations: in the adipose tissue dorsal lateral to the thymus, posterior to the trachea and where the trachea branches. When picking lymph nodes, cut the sternum with scissors, open the thoracic cavity left and right, and expose the left and right thymus glands above the heart. There are 3 or 4 thymic lymph nodes attached to the inner side of the left and right thymus, corresponding to the bronchial branches. The color of lymph nodes is similar to the white thymus, be careful when removing it.

5.  Inguinal lymph nodes

The inguinal lymph nodes are almost kidney-shaped, superficially in a lacuna deep in the left and right gluteus muscles, and the head is the origin of the sciatic nerve. Cut the muscle group of the lower back from the front end, peel off the superficial layer, and gradually lift it up to the end to find the pair of lymph nodes. Due to the small size of this pair of lymph nodes, it is hard to find them again if their local relationship is accidentally disturbed.

6.  Pancreatic lymph nodes

The pancreatic lymph nodes are a close-fitting, nearly spherical pair, with abdominal blood vessels passing through the middle, between the medial borders of the left and right kidneys and the abdominal aorta, near the caudal end of the adrenal glands. Turn the kidneys inward and peel off fat tissue to obtain the renal hilar lymph nodes without damaging the large intra-abdominal blood vessels. During extraction, the left lymph node is attached to the ventral side of the renal artery and renal vein and can be easily extracted. While the right lymph node is located at the back of the renal artery and renal vein, and it is difficult to remove if it is not visible. Therefore, the right kidney can be turned toward the direction of left kidney, and the right lymph node can be seen attached near the abdominal aorta, which can be easily removed.

7.  Mesenteric lymph nodes

This is the largest and easiest to find lymph node in all lymph nodes of the mouse. It is shaped like an earthworm and extends into the adipose tissue of the mesentery. During extraction, find the cecum first, trace up the mesentery and cut the mesenteric tissue then remove the lymph nodes.

8.  Lumbar lymph nodes

The lumbar lymph nodes are located on both sides of the abdominal aorta and are attached to the upper part of its bifurcation.

9.  Tail lymph nodes

The shape is nearly spherical, located on the right side of the branch of the abdominal aorta. The volume of Tail lymph nodes is relatively small, so look for it carefully.

10. Lymph nodes in stomach

There may be some changes in its location. Most of the lymph nodes can be found in the lesser curvature, and occasionally two lymph nodes can be found in the cardia or the lower end of the esophagus.

11. Axillary lymph nodes

The axillary lymph nodes are nearly spherical, located in the superficial subcutaneous adipose tissue of the left and right armpits and easy to find. However, the skin should not be separated from the local part during dissection, otherwise the anatomical relationship will be disturbed and it will be difficult to find.

Number and diameter of lymph nodes in mouse

1.   Number of lymph nodes

Mostly only one lymph node can be seen in parts of mouse body. However, 2~3 lymph nodes can be seen in the superficial neck, internal chest, stomach, pancreas and mesentery, and even as many as 4 lymph nodes. Among these lymph nodes, the number of intrathoracic lymph nodes changes more often. The probability of dissection of lymph nodes is different in different parts. For example, lymph nodes in the deep neck, tail or ischium, these lymph nodes are difficult to find and require more skilled anatomical techniques and careful operation. While finding lymph nodes in the superficial neck, axilla or mesentery, obtaining lymph nodes is more likely to succeed.

2.   Long diameter of lymph nodes

Most of long diameter of the lymph nodes is between 2 and 3.5 mm. The largest is mesenteric lymph node, whose long diameter is more than 10 mm, and the smallest is the deep neck, ischial and caudal lymph nodes, all of which are equal to or less than 1 mm in long diameter. Generally, normal lymph nodes do not exceed 4 mm in length and diameter.