For the preparation of mouse spleen cells we recommend cutting the spleen once and passing the tissue through a 100µM cell strainer or steel mesh using a plunger. The cells are flushed into a petri dish containing PBS. In order to remove fat, cell debris and aggregates, the cells can further be passed through a 70µM cell strainer or a cotton wool column. With this procedure, each spleen should yield approximately 50 million-200 million cells. We strongly recommend omitting the erythrocyte lysis step since it leads to a decrease in lymphocyte viability after Nucleofection®. Due to relatively low numbers of erythrocytes in the spleen cell preparation (1:2 erythrocytes: leukocytes compared to 1:1000 in human peripheral blood), an erythrocyte lysis is not required.