Mouse Spleen Cell Isolation Protocol

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.

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• 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.

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