小鼠脾脏细胞分离Protocol Mouse Spleen Cell Isolation Protocol
时间：2024-06-20 阅读：331
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. |
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. |