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小鼠脾臟細胞分離Protocol Mouse Spleen Cell Isolation Protocol

更新時(shí)間:2024-06-20   點(diǎn)擊次數:158次

脾臟是造血、紅細胞清除和免疫功能的場(chǎng)所,因此是細胞質(zhì)控的良好來(lái)源。它可以過(guò)濾細胞碎片、病原體和不規則細胞。它是紅細胞和白細胞以及幾種免疫細胞亞型的來(lái)源,包括粒細胞、單核細胞、巨噬細胞、樹(shù)突狀細胞 (DC)、NK 細胞、T 細胞和 B 細胞。


小鼠脾臟細胞分離Protocol Mouse Spleen Cell Isolation Protocol

操作步驟

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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