TITLE: How to Prepare a Single Cell Suspension from Mouse Spleen
AUTHORS: STEMCELL Technologies

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The spleen is an important organ of the immune system responsible for filtering blood and initiating immune responses to blood-borne antigens. Various blood, lymphoid, and hematopoietic cell types may be isolated from spleen samples for further study of the immune system. Preparing a true 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. This protocol describes how to harvest cells from a spleen sample, and prepare a single cell suspension prior to performing cell isolation.</div></div>

[STEPS]
?. [Mechanical Digestion of a 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. If you are not using an isolation kit following dissociation please use phosphate buffered saline (PBS) + 1 mM EDTA.Recommended Medium:
Note: If processing multiple spleens please use a 100 mm culture dish containing 10 mL of recommended medium.
?. [Mechanical Digestion of a Spleen Sample]
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, discolouration, 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.
?. [Mechanical Digestion of a Spleen Sample]
Take the piston/plunger out of a sterile 3 cc syringe. Use the flat back end of this device 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.
?. [Prepare a Single Cell Suspension]
Over a sterile 50 mL conical tube, place a 70 μm cell strainer and pass 2 mL of recommended medium through to prime the filter.
Note: Priming the filter reduces cell adhesion that may occur if the filter is dry when cells are passed through.
?. [Prepare a Single Cell Suspension]
Triturate the released splenocytes to a homogeneous mixture with a primed 5 or 10 mL serological pipette.
?. [Prepare a Single Cell Suspension]
Transfer the supernatant and tissue from the culture dish to the primed 70 μm cell strainer. Using a fresh piston/plunger out of a sterile 3 cc syringe gently pass the cells and the dissociated tissue through the cell strainer membrane by pressing in a circular motion with the plunger/piston end of the syringe. Wash the strainer with 3 mL of recommended medium. Repeat this step and then discard the leftover tissue and strainer.
?. [Prepare a Single Cell Suspension]
Top up the tube containing the cell suspension with PBS, and collect the cells by centrifugation of the tube at
?. [Prepare a Single Cell Suspension]
Carefully discard the supernatant without disturbing the pellet.
?. [Prepare a Single Cell Suspension]
Gently tap the tube to resuspend cells in the residual volume.Splenocytes are now ready for downstream use.
Optional: A second wash step can be performed at this stage.