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小鼠脾脏细胞分离Protocol Mouse Spleen Cell Isolation Protocol

更新时间:2024-06-20   点击次数:210次

脾脏是造血、红细胞清除和免疫功能的场所,因此是细胞质控的良好来源。它可以过滤细胞碎片、病原体和不规则细胞。它是红细胞和白细胞以及几种免疫细胞亚型的来源,包括粒细胞、单核细胞、巨噬细胞、树突状细胞 (DC)、NK 细胞、T 细胞和 B 细胞。


小鼠脾脏细胞分离Protocol Mouse Spleen Cell Isolation Protocol

操作步骤

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

1. Obtain fresh whole mouse spleen.

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

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

4. 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.

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

6. Place cell strainer over a 50 mL conical tube.

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

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

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

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

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

12. Incubate the suspension for 5 minutes on ice.

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

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

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




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