Mouse Ascites and Single Cell Suspension Preparation Process and Precautions

Preparation process of mouse ascites and single cell suspension

1)   Prepare 6% starch broth.

Preparation method: 0.3 g beef extract, 1.0 g peptone, 0.5 g sodium chloride, 100 mL distilled water, mix and heat the above materials, then add 6.0 g soluble starch into the mixture. After dissolving, autoclave the solution at 121 °C for 15~20 min. The starch broth were packaged and sealed with EP tubes and stored at 4°C.

2)   Inject 1 mL of 6% starch broth into abdominal cavity of mouse (do not touch intestinal tubes and internal organs) and stimulate for 60~72 h.

3)   Kill mouse by cervical dislocation and soak it in 75% alcohol for 5 min.

4)   Place the mouse in the dissection plate, fix the limbs and cut the skin to fully expose the peritoneum.

5)   Lift the peritoneum with ophthalmic tweezers, inject 2.5 mL of pre-cooled PBS into mouse abdominal cavity with a 5 mL syringe (do not puncture the organs), and gently rub mouse abdomen for 1~2 min. Withdraw the peritoneal lavage fluid with a syringe and collect in a 15 mL centrifuge tube.

6)   Repeat step 5 for 5 times, and it can be observed that the flushing fluid gradually becomes clear.

7)   Centrifuge the collected peritoneal lavage fluid at 300 g for 5 min, and discard the supernatant.

8)   Resuspend cells in Cell staining buffer or 1640 medium containing 10% fetal bovine serum.

9)   Count the cells and adjust the cell concentration to 1×10⁷/mL.

FSC/SSC diagram of mouse ascites cells

Precautions:

1. If there are many red blood cells in the cell pellet after centrifuging the supernatant in step 7, and the target cells are not red blood cells, add an appropriate amount of red blood cell lysate to lyse the red blood cells (you can add 500 μL of 1× red blood cell lysate to resuspend the cells at room temperature. After lysis for 2 min, add 10 mL of PBS immediately, and discard supernatant after centrifugation at 300 g for 5 min).

2. Please pay attention to aseptic operation if you need to extract and culture peritoneal macrophages.

3. When collecting ascites, inject PBS from the left side of the mouse (more intestines), and draw peritoneal lavage fluid from the right side (larger organs). Take care not to puncture the organs.

4. The cells collected from the peritoneal lavage fluid of mouse are fragile. Adding cell staining buffer is beneficial to the preservation of cells, and it is recommended to detect in time on the same day.

5. The majority cells in mouse peritoneal lavages are macrophages, and the detection of macrophages needs to block Fc receptors. Pure mouse CD16/32 antibody [E-AB-F0997A] can be used for blocking: add 1 μg of pure antibody to the 100 μL (Number of cells 1 × 10⁶) cell suspension, block at room temperature for 15 min, and directly add flow cytometry antibody for subsequent experiments.

6. For detection of macrophage, the use of anthocyanin-containing flow cytometry antibodies (such as PE-Cy7) should be avoided, otherwise non-specific staining will increase. For detection of non-macrophage, blocking is also required when using anthocyanin-containing flow cytometry antibodies such as PE-Cy7.