Preparation of mouse ascites and single cell suspension and precautions

Mouse ascites and single cell suspension preparation procedure
1. Prepare 6% starch broth.
6% starch broth preparation method: beef paste 0.3 g, peptone 1.0 g, sodium chloride 0.5 g, distilled water 100 mL, the above materials are mixed and heated to add 6.0 g of soluble starch; after dissolution, autoclave at 121 ℃ for 15~20 min, EP tubes are divided and sealed, and the broth is placed at 4 ℃ for storage.
2. Mice were injected intraperitoneally with 1 mL of 6% starch broth (taking care to avoid intestinal tubes and internal organs), and stimulated for 60~72 h. The mice were then injected with 1 mL of 6% starch broth.
3. Mice were executed by cervical dislocation and soaked in 75% alcohol for 5 min.
4. Place the mice in the dissecting plate, fix the limbs, cut the skin, and fully expose the peritoneum.
5. Lift the peritoneum with ophthalmic forceps, inject 2.5 mL of pre-cooled PBS into the peritoneal cavity of the mouse with a 5 mL syringe (do not puncture the organs), and gently knead the abdomen of the mouse for 1~2 min. withdraw the peritoneal lavage fluid with the syringe and collect it in a 15 mL centrifuge tube. 6. Repeat step 5 five times.
6. Repeat step 5 five times to rinse the abdominal cavity, and the rinsate is gradually clarified.
7. Centrifuge the collected lavage fluid at 300 g for 5 min and discard the supernatant. 8.
8. Resuspend the cells with cell staining buffer or 1640 culture medium containing 10% fetal bovine serum.
9. Perform a cell count and adjust the cell concentration to 1×107/mL.

FSC/SSC diagram of mouse ascites

Notes:
1. If more erythrocytes are found in the cell sediment after centrifugation and discarding the supernatant in step 7, and the target cells of the assay are not erythrocytes, add an appropriate amount of erythrocyte lysate to lysed the erythrocytes first (500 μL of 1× erythrocyte lysate can be added to re-suspend the cells, and after lysing the cells at room temperature for 2 min, immediately add 10 mL of PBS, centrifuge at 300 g for 5 min, and then discard the supernatant).
2. If the extracted peritoneal macrophages need to be cultured, please pay attention to the aseptic operation.
3. When collecting ascites, inject PBS from the left side of the mouse (more intestinal), and draw peritoneal lavage fluid from the right side (larger organs), taking care not to puncture the organs.
4. Mouse peritoneal lavage fluid collection cells are more fragile, plus cell staining buffer is conducive to the preservation of cells, but it is recommended that the same day timely detection.
5. Most of the cells in the mouse peritoneal lavage fluid are macrophages, and macrophage detection requires Fc receptor blocking, in this case, the mouse CD16/32 pure antibody [E-AB-F0997A] can be used for blocking: add 1 μg of pure antibody to 100 μL of cell suspension (cell number 1×106), block it for 15 minutes at room temperature, and then add the streaming antibody to carry out the subsequent experiments directly.
6. For macrophage detection, try to avoid the use of anthocyanin-containing flow antibodies (e.g., PE-Cy7), which will increase non-specific staining. For non-macrophage assays, the use of anthocyanin-containing flow-through antibodies (e.g., PE-Cy7) also requires containment.

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