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.
- 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)
Perform steps 1–7 at room temperature and steps 8–12 on ice with cold buffers.
For Research Use Only. Not for use in diagnostic procedures.