Hello,

Here the procedure

1. Harvest mouse spleen and prepare a single cell suspension.

2. Pellet the cells by centrifugation (350 x g); aspirate the supernatant.

3. Dilute the 10X Red Blood Cell Lysis Buffer to 1X working concentration with deionized water and resuspend the pellet in 3-5 ml of 1X Lysis Buffer.

4. Incubate on ice for 4-5 minutes with occasional shaking.

5. Stop the reaction by diluting the Lysis Buffer with 10-20 ml of 1X PBS.

6. Spin the cells (350 x g), discard the supernatant, and resuspend the pellet in the appropriate buffer

7. Count cells, adjust density, and proceed with cell staining procedures.

Perform lysis the spleenocytes first and then freeze with cell freezing solution (90% FBS + 10% DMSO ).

You can put spllen in complete media overnight and then perform lysis tomorrow. If you can`t control to collect samples, you can try freeze some pieces of spleen in OCT solution with 20%sucrose like frozen samples.

Hope this info will help you.

Good luck!

Thanks.

Your welcome!