1) Sacrifice the mice and bleed them by aortic section.
2) Pin the mice to a dissecting board with the left-side up.
3) Wash the mouse with 70% ethanol and make a longitudinal incision exposing the peritoneal cavity.
4) Remove the spleen by lifting it at one end and cutting away from the body.
5) Place the spleen on a sterile mesh.
6) Press the spleen through the mesh using a sterile syringe plunger and collect the cells in a perti dish containing 5 ml of cold RPMI 1640 (Invitrogen).
7) Wash the cell suspension 5 times with RPMI 1640 by pelleting cells at 300 g for 15 minutes at 4 C using a bench-top refrigerated centrifuge.
8) Resuspend the cell pellets in 5 ml HBSS for separation by Percoll™ density gradient centrifugation. See here for the protocol. http://clodrosome.blogspot.com/2012/01/percoll-continuous-density-gradient-for.html
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