Introduction

The spleen is the site for hematopoiesis, red blood cell clearance, and immunologic functions, and therefore, a good source of cells. It filters cell debris, pathogens, and irregular cells. It is a source for both red blood cells and leukocytes and for several immune cell subtypes including granulocytes, monocytes, macrophages, dendritic cells (DCs), NK cells, T cells, and B cells. Leukocytes can be found in the crude spleen preparation. DCs and macrophages can be isolated by enzymatic release of the cells from the crude cell preparation.


Materials


Preparation of myeloid cells


Procedure

Perform steps 1–7 at room temperature and steps 8–12 on ice with cold buffers.

  1. Obtain fresh whole mouse spleen.
  1. Place mouse spleen into petri dish with 5 mL HBSS (Hank’s balanced salt solution) buffer.
  1. Carefully mince the spleen into small pieces (~0.2 cm2) with a razor or scalpel blade.
  1. For preparation of myeloid cells (continue to step 5 for crude preparation): Incubate the excised spleen pieces for 20–30 min at 37°C with 5 mL of HBSS containing Collagenase IV (100 U/mL) and DNase (20 µg/mL) solution with 1% FBS.

    4a. Add EDTA 1 mM/mL for 5 minutes at room temperature to stop the enzymatic reaction. 
  1. Place cell strainer over a 50 mL conical tube.
  1. With a disposable transfer pipette, transfer the excised spleen into the cell strainer.
  1. With the plunger end of a syringe, mash or press the spleen through the strainer. Add 5–10 mL PBS if necessary.
  1. Wash the cells through the strainer with excess PBS. Repeat step 5 and 6, if needed.
  1. Centrifuge the cells at 400–600 x g for 5 minutes at 4°C; discard the supernatant.
  1. Resuspend the cell pellet in 2–5 mL of cold 1x RBC Lysis buffer.
  1. Incubate the suspension for 5 minutes on ice.
  1. Wash the cell suspension with 10–20 mL cold PBS.
  1. Centrifuge the cells at 400–600 x g for 5 minutes at 4 °C; discard the supernatant.
  1. Resuspend the cell pellet in PBS at 2–3 x 106 cells/mL.