Mouse Thymus Single-Cell Suspension Preparation

I. Materials Preparation

1. Surgical Instruments: Including ophthalmic scissors, forceps, etc.

2. Culture Dish: Disposable plastic culture dish.

3. Cell Filter: 70 μm or 200 mesh cell filter to separate cell clumps and ensure the preparation of a single-cell suspension.

4. Centrifuge Tubes: 15 mL or 50 mL centrifuge tubes for collecting and centrifuging cells.

5. PBS or Culture Medium: For washing cells.

Note: If the cells are to be cultured afterwards, sterilise the materials to ensure aseptic conditions.

II. Harvesting Mouse Thymus

Euthanise the mouse using cervical dislocation, then immerse in 75% ethanol for 5 minutes. Place the mouse on the operating surface. Using scissors and forceps, make a small incision at the sternum, tear the skin to expose the sternum. Cut the sternum and ribs on both sides of the heart, then carefully remove the thymus from the anterior mediastinum above the heart, and place it in 1×PBS buffer solution.

Figure 1. Location of Mouse Thymus (Green Box)

III. Preparation of Thymus Single-Cell Suspension

1. Using the flat end of a 5 mL syringe plunger or a pestle, gently grind the thymus in a circular motion until no obvious red clumps are visible.
2. Filter the cells through a 270 μm or 200 mesh filter, then wash the filter with an appropriate amount of PBS or culture medium. Collect the cells in a centrifuge tube. Centrifuge at 300-500 g for 5 minutes and discard the supernatant.
3. Resuspend the cells in PBS or culture medium, count the cells, and adjust the concentration to 1×10⁷/mL.

Figure 2. Flow Cytometry FSC/SSC Results of Mouse Thymus Cells

IV. Notes

1. When opening the thoracic cavity, be careful not to damage the heart or major blood vessels.

2. Apply gentle grinding pressure to avoid excessive mechanical cell death.

3. If the thymus cells are to be cultured further, ensure aseptic handling; if they are only used for ordinary flow cytometry experiments, sterile conditions are not essential.

4. Cell counting can be performed using a haemocytometer or an automated cell counter.

5. To maintain cell viability and reduce metabolic rate, keep the samples at low temperatures throughout the procedure (on ice or in a 4°C environment).

abinScience Flow Cytometry (FACS) Antibodies