Mouse Naïve CD4 T Cells Isolation

1. Mouse Naïve CD4+ T Cells Isolation 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 https://www.creative-diagnostics.com/mouse-naive-cd4-t-cells-isolation.htm