Mouse Spleen Single Cell Suspension Preparation Process and Precautions

Preparation process of mouse spleen single cell suspension

a) Grinding method

1)    Kill mouse by cervical dislocation and soak it in 75% alcohol for 5 min, then place the mouse on a sterile operating table with the left ventral side up.

2)    Cut a small incision in the middle of the left ventral side of the mouse, torn the skin open and expose the abdominal wall, a long red spleen will be visible.

3)    Lift the peritoneum from the lower side of the spleen, cut it open and turn it up to expose the spleen. Lift the spleen with forceps, separate the connective tissue below the spleen with ophthalmic scissors, remove the spleen, and soak it in clean PBS solution.

4)    Place the spleen in a 200-mesh sieve and grind gently with a tissue grinder until there are no obvious red lumps.

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

6)    Add 2 mL of 1× erythrocyte lysate to resuspend the cells. After lysing at room temperature for 2~3 minutes, immediately add 10 mL of PBS. Centrifuge the solution at 300 g for 5 minutes, discard the supernatant.

7)    Resuspend the spleen cells with cell staining buffer, filter the cell suspension again with a 200-mesh sieve and count, and adjust the cell concentration to 1×10⁷/mL.

b) Blowing method

1)    Kill mouse by cervical dislocation and soak it in 75% alcohol for 5 min, then place the mouse on a sterile operating table with the left ventral side up.

2)    Cut a small incision in the middle of the left ventral side of the mouse, torn the skin open and expose the abdominal wall, a long red spleen will be visible.

3)    Lift the peritoneum from the lower side of the spleen, cut it open and turn it up to expose the spleen. Lift the spleen with forceps, separate the connective tissue below the spleen with ophthalmic scissors, remove the spleen, and soak it in clean PBS solution.

4)    Take a 2.5 mL sterile syringe to suck PBS, hold the spleen with tweezers in the left hand and the syringe in the right hand, carefully insert PBS into the spleen and pipette until the spleen cells are completely cleaned, and observe that only white connective tissue and adipose tissue remain. Pick up the remaining white tissue with tweezers and rinse gently in PBS.

5)    Filter the pipetted cells with a 200-mesh sieve, collect them in a 15 mL centrifuge tube, centrifuge at 300 g for 5 min, and discard the supernatant.

6)    Add 2 mL of 1× red blood cell lysate to resuspend the cells, lyse at room temperature for 2~3 minutes and immediately add 10 mL of PBS. Centrifuge the solution at 300 g for 5 minutes, and discard the supernatant.

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

FSC/SSC diagram of mouse spleen cells

Precautions:

1.  For a normal-sized spleen, about 4×10⁷ cells can be obtained according to previous experience. The actual number of cells is subject to the counting result.

2.  Lymphocytes account for about 60%~70% of the total cells in mouse spleen after lysing red blood cells.

3.  For unstained spleen cell samples, a small amount (about tenths of a percent) of non-specific signal can be seen in the fluorescence channel.

4.  If there is no tissue grinding rod, the plunger inside the syringe can also be used instead. Grind the spleen with a rubber pad on the tip of the pusher.

5.  If the collected spleen cells need to be further cultured, the mouse spleen should be placed on a sterile operating table. If you are just doing ordinary flow cytometry experiments, you can ignore the sterile environment.

6.  PBS can be replaced with Cell Staining Buffer.

7.  In the step of lysing red blood cells, the lysis time is determined by the effect of fission during the experiment.