This protocol describes how to harvest cells from a mouse spleen sample and how to prepare a single-cell suspension of splenocytes prior to performing cell isolation. Preparing a single-cell suspension of the primary tissue sample will optimize cell separation by avoiding additional cell loss and enabling maximum labeling of the target cells.
Part I: Mechanical Digestion of a Mouse Spleen Sample
Transfer the spleen to be processed into a sterile 35 mm culture dish containing 5 mL of recommended dissociation medium for downstream isolation (this will be indicated in your cell isolation kit's Product Information Sheet). If you are not using an isolation kit following dissociation, use phosphate-buffered saline (PBS) + 1 mM EDTA.
Note: If processing multiple spleens, use a 100 mm culture dish (e.g. Catalog #38045) containing 10 mL of recommended dissociation medium.
Trim any extra connective tissues or fat from the spleen, take note of any necrotic regions, and inspect the spleen for any other phenomena such as enlargement, discoloration, or lesions. It is important to note the condition of your starting sample, as this may be taken into consideration when evaluating your cellular fraction.
Remove the piston/plunger from a sterile 3 cc syringe. Use the flat end of the plunger to mince the spleen by crushing the spleen 5 times in gentle circular motions. This action will burst the spleen, disrupt the pulp, and release the splenocytes.
Part II: Preparation of a Single-Cell Suspension
Place a 70 µm cell strainer on a sterile 50 mL conical tube. Pass 2 mL of recommended medium through the strainer to prime it.
Note: Priming the strainer reduces cell adhesion that may occur if the strainer is dry when cells are passed through.
Triturate the released splenocytes to a homogeneous mixture with a primed 5 or 10 mL serological pipette.
Transfer the supernatant and tissue from the culture dish to the primed strainer. Using a fresh piston/plunger from a sterile 3 cc syringe, gently pass the cells and the dissociated tissue through the strainer by pressing in a circular motion with the flat end. Wash the strainer with 3 mL of recommended medium. Repeat this step and then discard the leftover tissue and strainer.
Top up the tube containing the cell suspension with PBS. Centrifuge the tube at 300 x g for 10 minutes.
Carefully remove and discard the supernatant without disturbing the pellet.
Gently tap the tube to resuspend cells in the residual volume. An optional second wash can be performed at this stage. Splenocytes are now ready for downstream use.
Isolate Mouse Cells in as Little as 15 Minutes
Use EasySep™ to obtain highly purified mouse immune cells from various sample sources, including mouse splenocytes.