Protocol for Mouse spleen isolation and preparation of single-cell suspension

Before carrying out any lab procedures, please consult your lab protocol and safety guidelines. Also, ensure all equipment used has been cleaned and sterilized according to your lab’s protocols. This is a simplified protocol, and it should be adjusted to suit specific experimental parameters:

Materials needed:

– Sterile surgical instruments (scissors, forceps)

– 70 μm cell strainer

– 50 ml centrifuge tube

– PBS (Phosphate-buffered saline)

– DMEM (Dulbecco’s Modified Eagle Medium) or similar culture medium

– Fetal Bovine Serum

– Penicillin/Streptomycin or suitable antibiotic

– 5% CO2 incubator

– Centrifuge

– Water bath

-Hank’s Balanced Salt Solution (HBSS)

Procedure:

1. Sterilize your work area and all necessary materials.

2. Euthanize the mouse following your institution’s approved methods for animal handling.

3. Soak the animal with 70% ethanol to reduce the risk of contamination, and use dissecting scissors to expose the abdominal cavity.

4. Identify the spleen, a small red organ next to the stomach. Carefully collect the spleen using sterile scissors and forceps.

5. Place the spleen in a sterile petri dish containing cold, sterile PBS.

6. Cut the spleen into small pieces using sterile forceps and scissors.

7. Transfer the spleen fragments to a 70 μm cell strainer placed over a 50 ml centrifuge tube.

8. Gently disrupt the spleen fragments with a syringe plunger to push the cell suspension through the strainer into the tube. Rinse with PBS.

9. Centrifuge the cell suspension at 300 g for 10 minutes at 4°C.

10. Discard the supernatant and resuspend the pellet in red blood cell lysis buffer (if necessary) to remove any red blood cells. Incubate for a few minutes at room temperature.

11. Add 10 times the volume of PBS to stop the lysis reaction.

12. Centrifuge again at 300 g for 10 minutes at 4°C to pellet the cells.

13. Discard the supernatant and gently resuspend the pellet in an appropriate PBS or culture medium volume.

14. Count the cells using a hemocytometer or automated cell counter.

Your single-cell suspension from the mouse spleen is now ready to be used for subsequent experiments. If not used immediately, cells can be stored briefly on ice at 4°C or cryopreserved (liquid nitrogen) for longer storage (cells cryopreserved using 10% DMSO + 90% FBS /mL in a cryo vial).

Remember: Some experimental protocols may additionally require cell sorting or other post-isolation procedures. Always follow your specific lab instructions and protocols.