Mouse Spleen Cell Isolation Protocol | Thermo Fisher Scientific

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

Red blood cell (RBC) lysis buffer (e.g., eBioscience 10X RBC Lysis Buffer (Multi-species), Cat. No. 00-4300-54)
Phosphate buffer saline (e.g., Gibco PBS (10X), pH 7.4, Cat. No. 70011044)
Scalpel and blades
Cell culture dishes (e.g., Thermo Scientific Nunc EasYDish Dishes, 100 mm, Cat. No. 150466)
Disposable transfer pipette (e.g., FisherBrand Disposable Graduated Transfer Pipettes, Cat. No. 137112)
50 mL conical tubes (e.g., Thermo Scientific Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes, Cat. No. 339652)
70 μm cell strainers (e.g., Fisherbrand Sterile Cell Strainers, Cat. No. 22-363-548)
5–10 mL syringe (e.g., Fisherbrand Sterile Syringes for Single Use, 10 mL, Cat. No. 14-955-459)
Hank’s balanced salt solution (e.g., Gibco Hank’s Balanced Salt Solution, HBSS 10x, Cat. No. 14060040)

Preparation of myeloid cells

Collagenase (e.g., Gibco Collagenase, Type IV, Cat. No. 17104019)
DNase (e.g., Thermo Scientific DNase I solution, Cat. No. 90083)
Fetal Bovine Serum (e.g., Gibco Fetal Bovine Serum, Cat. No. 26140087, or Fetal Bovine Serum One Shot format, Cat No. A3160401)
EDTA (e.g., UltraPure 0.5M EDTA, pH 8.0, Cat. No. 15575020)

Procedure

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

Obtain fresh whole mouse spleen.

Place mouse spleen into petri dish with 5 mL HBSS (Hank’s balanced salt solution) buffer.

Carefully mince the spleen into small pieces (~0.2 cm²) with a razor or scalpel blade.

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 solution containing Collagenase IV (100 U/mL), DNase I (20 U/mL), and 1% FBS.

4a. Add EDTA to the solution to a concentration of 1 mM for 5 minutes at room temperature to stop the enzymatic reaction.

Place cell strainer over a 50 mL conical tube.

With a disposable transfer pipette, transfer the excised spleen into the cell strainer.

With the plunger end of a syringe, mash or press the spleen through the strainer. Add 5–10 mL PBS if necessary.

Wash the cells through the strainer with excess PBS. Repeat step 5 and 6, if needed.

Centrifuge the cells at 400–600 x g for 5 minutes at 4 °C; discard the supernatant.

Resuspend the cell pellet in 2–5 mL of cold 1x RBC Lysis buffer.

Incubate the suspension for 5 minutes on ice.

Wash the cell suspension with 10–20 mL cold PBS.

Centrifuge the cells at 400–600 x g for 5 minutes at 4 °C; discard the supernatant.

Resuspend the cell pellet in PBS at 2–3 x 10⁶ cells/mL.

For Research Use Only. Not for use in diagnostic procedures.