Mouse Thymus Single Cell Suspension Preparation Process and Precautions

Preparation process of mouse thymus single cell suspension

1)    Kill mouse by cervical dislocation and soak it in 75% alcohol for 5 min. Place the mouse in the sterile operating table with belly up.

2)    Cut the thoracic cavity below the sternum of the mouse, and the white transparent thymus can be seen. The thymus are distributed in two lobes and located in front of the two lungs, just behind the sternum.

3)    Remove the thymus and soak it in clean PBS solution.

4)    Place the thymus in a 200-mesh sieve and lightly grind it with a tissue grinder until there are no obvious lumps.

5)    Rinse the mesh with 15 mL of PBS and collect the rinse solution in a 15 mL centrifuge tube. Centrifuge the solution at 300 g for 5 min, and discard the supernatant.

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

FSC/SSC diagram of Mouse thymus cells

Precautions:

1.  Open the thoracic cavity to separate the thymus, taking care not to cut off the great vessels and break the heart.

2.  If necessary, inject 0.1 mL of black ink into abdominal cavity before the mouse is executed to stain the lymph nodes in the thoracic cavity. This step will facilitate lymph nodes identification and removal.

3.  Generally, 2× 10⁸ cells can be obtained from 3~6 week old mice. Thymocytes cell number gradually decreases as mice age.