Mouse Spleen Cell Isolation Protocol | Thermo Fisher Scientific

Introduction

This mouse spleen cell isolation protocol outlines the steps for isolating splenocytes, which include various immune cell subtypes such as granulocytes, monocytes, macrophages, dendritic cells (DCs), NK cells, T cells, and B cells. Splenocyte isolation involves processing the spleen tissue to release cells while filtering out cell debris, pathogens, and irregular cells. The spleen is a critical organ for hematopoiesis, red blood cell clearance, and immunologic functions, making it an excellent source of diverse cells. It helps provide a rich source of both red blood cells and leukocytes, with leukocytes found in crude spleen preparations and DCs and macrophages isolated through enzymatic release. This mouse splenocyte isolation protocol helps ensure a reliable method for spleen cell isolation, facilitating detailed analysis using evaluation methods such as flow cytometry, ELISA, and cell culture assays for various research applications.

Search mouse cell isolation dynabeads Search mouse primary antibodies

On this page:

Materials
Isolation procedure
Ordering information

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.

Ordering information

Isolation materials

Preparation of myeloid cells

Top

Resources

Immunology at Work Resource Center
Find guides and protocols for immunology research.

Protocols for Immunology
Discover protocols and eLearning courses for various applications to study immunology.

Immune Cell Guide
Download this guide that provides detailed marker information for immune cell types and subtypes.

Cell Line Information and Supporting Products
Get information for splenocytes and other cell lines along with product recommendations and associated applications.