1. Decant the spleen into the sterile glass Petri dish and gently dissociate by rubbing between the frosted ends of the glass microscope slides. It is sometimes necessary to break up the spleen a little before dissociating using either the ends of the microscope slides or a pair of dissecting scissors. After dissociation there will be fibrous tissue remaining from the spleen capsule and a red liquid containing the splenocytes.
2. Aspirate the red liquid from the fibrous tissue and place into a sterile universal container. Allow the contents of the universal container to settle for a few minutes then aspirate or "pour off" the supernatant and retain it, as this contains the splenocyte (see Note 10).
3. Wash the splenocytes with PBS by centrifugation at 400g and resuspend the pellet in 10 mL of cold PBS.
4. Remove 2.5 mL of splenocyte suspension for cell fusion.
5. Harvest the remaining cells by centrifugation, resuspend in freezing medium (2 mL/spleen) and dispense into cryotubes in 0.5-mL aliquots. Freeze the cells and store cryogenically.
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