Materials Required:

• Sterile oral swabs (cotton-tipped or specialized brushes)
• Sterile PBS (phosphate-buffered saline)
• Centrifuge tubes (typically 1.5 ml or 2 ml tubes)
• Cell culture medium (such as DMEM/F12 or other suitable medium for epithelial cells)
• Pipettes and micropipettes
• Cell counting reagents (such as Trypan Blue)

General Procedure:

1. Anesthesia of Mice:

• Anesthetize the mice using an appropriate anesthetic, such as isoflurane or other suitable agents. Ensure that the mice are fully anesthetized to avoid pain and distress during the procedure.Inhalational anesthesia (e.g., isoflurane) can be administered in an anesthesia chamber until the mice lose consciousness. Alternatively, injectable anesthetics (such as ketamine-xylazine) may be used, following standard dosage guidelines.
• Confirm the loss of reflexes to ensure the mice are sufficiently anesthetized before proceeding with the oral mucosal cell collection. Adhere to institutional ethical guidelines for animal handling.

2. Oral Mucosal Sampling:

• Using a sterile oral swab (cotton-tipped or specialized brush), gently insert the swab into the mouse's oral cavity. Be cautious to avoid excessive force, as this could harm the mucosal tissue.
• Scrape the epithelial cells from the mucosal surface, typically from the buccal mucosa (inner cheek) or hard palate. A gentle rotating motion can be used to collect cells. Scraping should be done lightly to avoid damage to the underlying tissues.
• Ensure to scrape long enough to collect a sufficient number of cells, but avoid excessive scraping to prevent trauma to the mucosa.

3. Cell Recovery:

• Place the swab into a sterile centrifuge tube containing sterile PBS.
• Gently pipette up and down to help the cells detach from the swab into the PBS. Alternatively, the tube can be gently shaken to assist in the release of the cells.

4. Cell Collection:

• Remove the swab and discard it to avoid further contamination.
• Allow the centrifuge tube to stand briefly at room temperature, allowing the cells to settle to the bottom of the tube.
• Use a pipette to carefully transfer the supernatant to a new centrifuge tube if necessary for further processing.

5. Centrifugation:

• Centrifuge the tube at 300-500 × g for 5-10 minutes to pellet the cells at the bottom of the tube.
• After centrifugation, carefully discard the supernatant and retain the cell pellet.

6. Cell Resuspension and Culture:

• Resuspend the cell pellet in an appropriate volume of cell culture medium (e.g., DMEM/F12 supplemented with fetal bovine serum).
• Gently pipette the cell suspension to ensure the cells are evenly dispersed.
• Perform a cell count and assess cell viability using methods such as Trypan Blue staining, where dead cells will take up the dye.

7. Cell Culture (Optional):

• If required for further analysis, transfer the cells to appropriate culture dishes. Cells can be cultured in collagen-coated plates to promote attachment, as epithelial cells generally require a surface for proper adhesion.
• Culture the cells in a humidified incubator at 37°C with 5% CO₂ and monitor their growth and confluence.

Notes:

• Careful handling is essential to minimize stress and potential harm to the animals. Rapid and gentle sampling minimizes tissue damage.
• Sterile techniques must be maintained throughout the procedure to prevent contamination.
• If higher yields of cells or purification of specific populations are required, additional methods (e.g., enzymatic dissociation) may be considered.
• After sampling, ensure the animal recovers fully from anesthesia and is observed for any signs of distress or complications.

By following this procedure, you can collect and isolate oral mucosal epithelial cells from live mice for subsequent analysis and experimentation.

above is my reccomandation i have experience of thousands of patients oral swab during covid 19 pandemic .u can try

Dear Jun Ma,

Thanks you so much for the detailed reply,

Michael

here is oral swabs used by my lab and experienced before

Thank you very much