2026.01.12
The spleen is an organ with hematopoietic, red blood cell clearance and immune functions, containing a large number of lymphocytes that account for 25% of the total lymphoid tissue in the body. Due to the small blood volume of mice, it is difficult to isolate a large number of lymphocytes from peripheral blood, so the spleen is often used as an excellent source of immune cells and lymphocytes. It filters out cell debris, pathogens and abnormal cells, and is a source of red blood cells, white blood cells and various immune cell subsets (including granulocytes, monocytes, macrophages, dendritic cells (DC), NK cells, T cells and B cells). White blood cells can be obtained by crude spleen cell isolation, in which cells release enzymes to digest tissues for DC and macrophage acquisition.
Lymphocytes are the smallest white blood cells produced by lymphoid organs (including lymph nodes, spleen and thymus). Mainly present in lymph fluid circulating in lymphatic vessels, they are important cellular components of the body's immune response function, the main executors of almost all immune functions of the lymphatic system, and the front-line "soldiers" against external infections and monitoring cell mutations in the body. Lymphocytes are a type of cell line with immune recognition function, which can be divided into T lymphocytes (T cells), B lymphocytes (B cells) and natural killer (NK) cells according to their different development, migration, surface molecules and functions.
Red Blood Cell (RBC) Lysis Buffer
Phosphate Buffered Saline (PBS), pH 7.4
Scalpel and blade
Cell culture dish (100 mm)
Disposable pipettes
50 mL conical tubes
70 μm cell strainer
5–10 mL syringes
Hanks' Balanced Salt Solution (HBSS)
Perform steps 1–7 at room temperature, and steps 8–12 on ice using cold buffers.
1. Obtain a fresh mouse spleen.
2. Place the mouse spleen into a culture dish containing 5 mL of HBSS.
3. Carefully cut the spleen into small pieces (~0.2 cm²) with a razor or scalpel blade.
4. For myeloid cell preparation (continue to step 5 for crude isolation): Digest the small spleen pieces with 5 mL of HBSS containing Collagenase Type IV (100 U/mL) and DNase I (20 µg/mL) supplemented with 1% FBS at 37°C for 20–30 minutes.
5. a. Add 1 mM/mL EDTA and incubate at room temperature for 5 minutes to terminate the enzymatic reaction.
6. Place a cell strainer over a 50 mL conical tube.
7. Transfer the digested spleen to the cell strainer using a disposable pipette.
8. Mash or crush the spleen with the plunger end of a syringe to allow it to pass through the strainer. Add 5–10 mL of PBS for rinsing if necessary.
9. Rinse the cells thoroughly with a large amount of PBS to pass through the strainer. Repeat steps 5 and 6 if needed.
10. Centrifuge the cells at 400–600 x g for 5 minutes at 4°C, then discard the supernatant.
11. Resuspend the cells in 2–5 mL of pre-cooled 1x RBC Lysis Buffer.
12. Incubate the resuspension on ice for 5 minutes.
13. Wash the cell suspension with 10–20 mL of ice-cold PBS.
14. Centrifuge the cells at 400–600 x g for 5 minutes at 4°C, then discard the supernatant.
15. Resuspend the cells in PBS to a final concentration of 2–3 x 10⁶ cells/mL.
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