2. Place mouse spleen into petri dish with 5 mL HBSS (Hank’s balanced salt solution) buffer.

3. Carefully mince the spleen into small pieces (~0.2 cm2) with a razor or scalpel blade.

4. For preparation of myeloid cells (continue to step 5 for crude preparation): Incubate the excised spleen pieces for 20-30 min at 37°C with 5 mL of HBSS solution containing Collagenase IV (100 U/mL), DNase I (20 U/mL), and 1% FBS.

5. Add EDTA to the solution to a concentration of 1 mM for 5 minutes at room temperature to stop the enzymatic reaction.

6. Place cell strainer over a 50 mL conical tube.

7. With a disposable transfer pipette, transfer the excised spleen into the cell strainer.

8. With the plunger end of a syringe, mash or press the spleen through the strainer. Add 5–10 mL PBS if necessary.

9. Wash the cells through the strainer with excess PBS. Repeat step 5 and 6, if needed.

10. Centrifuge the cells at 400–600 x g for 5 minutes at 4 °C; discard the supernatant.

11. Resuspend the cell pellet in 2–5 mL of cold 1x RBC Lysis buffer.

12. Incubate the suspension for 5 minutes on ice.

13. Wash the cell suspension with 10–20 mL cold PBS.

14. Centrifuge the cells at 400–600 x g for 5 minutes at 4 °C; discard the supernatant.

15. Resuspend the cell pellet in PBS at 2–3 x 106 cells/mL.