Reagents:
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-CD62LhiCD44- 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.
