I have isolated fresh PBMC from whole blood using Histopaque 1077 and seeded onto a 96 well plate and incubated for 16-20 hrs in CO2 incubator. Few cytokines & chemokines secreted from the untreated PBMC is too high to consider as blank value. I have used multiple donors' blood over multiple days and all of them showed significantly high background. Therefore, it is not a donor specific issue. The cell viability was >90% after isolation. Please suggest what could be possible reasons for freshly isolated PBMC to secrete such high level of cytokines.