How to Prepare a Single-Cell Suspension from Mouse Spleen

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.


Materials


Protocol

Part I: Mechanical Digestion of a Mouse Spleen Sample

  1. 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.
  2. 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.
  3. 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

  1. 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.
  2. Triturate the released splenocytes to a homogeneous mixture with a primed 5 or 10 mL serological pipette.
  3. 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.
  4. Top up the tube containing the cell suspension with PBS. Centrifuge the tube at 300 x g for 10 minutes.
  5. Carefully remove and discard the supernatant without disturbing the pellet.
  6. 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.

  • Document #PR00013
  • Version 1.0.0
  • May 2020


  • Related Resources

    EasySep™ cell separation magnets

    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.

    Learn More >

    Choosing a Cell Separation Method that Meets Your Research Needs

    Learn more about the cell separation methods outlined above to choose the best method for your application.

    Compare Methods >

    Contact STEMCELL Technologies

    Our Customer Service, Sales, and Product and Scientific Support departments in North America are available between 6 am and 5 pm Pacific Time (9 am and 8 pm Eastern Time). One of our representatives will be happy to help you by telephone or email. Please complete the form to contact us by email. A representative will get back to you shortly.
    •  

    StemCell Technologies Inc. and affiliates ("STEMCELL Technologies") does not share your email address with third parties. StemCell Technologies Inc. will use your email address to confirm your identity and send you newsletters, transaction-related emails, promotional and customer service emails in accordance with our privacy policy. You can change your email preferences at any time.