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Reagents:

• RPMI+: RPMI 1640 with 10% FBS.
• ACK buffer: 150 mM NH₄Cl, 10 mM KHCO₃ and 500 μM EDTA, sterile-filtered.
• FACS buffer: PBS with 1 mM EDTA (pH = 8) and 1% BSA, sterile-filtered.

Procedures:

Prepare for cell suspension

1. Isolate spleen (or lymph nodes) from 5- to 10-week-old mice, and place in 5 mL of RPMI+ in 6 cm dish.

2. Cover the tissue with a 2×2 square of sterile nylon mesh, gently smash the tissue against the mesh using the thumb side of a 5-mL syringe plunger.

3. Filtrate cell suspension with 40 μm cell strainer, wash the dish and cell strainer with 5 mL PRMI+, and collect cell suspension in a 15 mL tube.

4. Invert on ice with a few times, centrifuge the cells at 800 rpm for 5 min at 4°C, remove supernatant.

5. Add 2 mL ACK buffer per spleen, gently suspend for 1 min to lyse erythrocytes, and add 10 mL of RPMI+.
Note: For lymph node cells, do not require erythrocyte lysis.

6. Centrifuge at 800 rpm for 5 min at 4°C, remove supernatant.

7. Resuspend with 10 mL RPMI+ buffer, filtrate cells into a new 15 mL tube.

CD4 enrichment

8. Count the cells, prepare biotin-conjugated antibodies against CD8a, CD11b, CD45R/B220, CD49b/ITGA2 and TER-119/LY76 at 1 μg/mL for 1×10⁷ cells in 100 μL of FACS buffer.

9. Incubate for 15 min on ice, gently mix for once during the incubation.

10. Add 1 mL serum-free DMEM, centrifuge at 300 g for 7 min at 4°C, remove supernatant.

11. Prepare Streptavidin microbeads at 25 μL/mL in 100 μL of DMEM for 1 × 10⁷ cells, incubate for 30 min on ice, gently mix for once during the incubation.

12. Separate antibody-bound and unbound cells by magnetic cell sorting system.

Sorting

13. Separate fluorescent-conjugated antibodies directed against CD62L, CD44, CD25, and CD4 in 100 μL of FACS buffer for 1 × 10⁷ cells.

14. Incubate for 30 min at 4°C in the dark, gently mix for once during the incubation.

15. Wash the cells with 1 mL RIPM+, and centrifuge 800 rpm for 5 min at 4°C.

16. Collect labeled cells into a new tube that is compatible for the cell sorter, keep on ice and protected from light.

17. Add 1 mL RPMI+ into the collection tubes.

18. Sort the CD4⁺CD25^-CD62L^hiCD44^- population.

19. Wash the cells collected with RPMI+, and centrifuge 800 rpm for 5 min at 4°C.

20. Resuspend with RPMI+, count the cells and adjust to an appropriate concentration.

21. Execute cell culture or in vitro differentiation.

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