We are doing a tetramer staining on splenocytes and blood lymphocytes from vaccinated mice. Both spleen and blood samples are treated with ACK lysis buffer prior to staining to remove erythrocytes. We are also using the same cell number in the staining protocol.

However, in the blood samples we detect a high background, even in unvaccinated control mice, which is not observed in the spleen samples. What could be the reason for the high background in blood but not spleen and how could we prevent this?

Thanks in advance!