Immune memory assessment

Protocol for immune memory assessment

1. Collect spleens from the untreated or treated mice. Cut them into 1~2mm³ tissue blocks. Put them into a tissue grinder and add 1~2mL PBS. Rotate the stick and grind until homogenate. Add 10mL PBS to rinse the grinder. Cell suspension was collected and filtered through 200~300 mesh nylon mesh.

2. Centrifuge the cell suspension at 300g for 5 min, and discard the supernatant. Wash these cell suspension three times using PBS containing 0.1% FBS.

3. Dilute 10x RBC lyse solution (ACK buffer) with deionized water to 1x and place at room temperature. The abovementioned cells were resuspended in 3mL 1x ACK buffer and incubated at room temperature for 3-5 min.

4. Add 10mL PBS containing 0.1% FBS to stop RBC lysis. The cell suspension at 300g was centrifuged for 5 min, and the supernatant was discarded. Wash these cell suspension three times using PBS containing 0.1% FBS.

5. Cells were counted and prepared into 1x10⁷/mL suspension with PBS containing 0.1% FBS. 

6. Add 0.5-1mg of pure anti-mouse CD16/32 monoclonal antibody and incubate for 10 min at room temperature. Wash these cell suspension using PBS containing 0.1% FBS.

7. Stained with anti-CD3-FITC (Biolegend, Clone: 145-2C11, Catalog: 100306, Lot: B241616), anti-CD8-PerCP-Cy5.5 (eBioscience, Clone: 53-6.7, Catalog: E08300-1633), anti-CD62L-APC (eBioscience, Clone: MEL-14, Lot:4300018) and anti-CD44-PE (Biolegend, Clone:IM7, Cat:103007, Lot: B185651) antibodies according to the manufacturers’ protocols.

8. Flow cytometry was used for analyzing the percentage of Central memory T cells (TCM, CD3 + CD8 + CD62L+CD44 +) and effector memory T cells (TEM, CD3 +CD8 +CD62L-CD44 +).

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