Cytokines, chemokines, and interferons are released by the immune cells in 
response to cellular stress, damage and/or pathogens, and are widely used as 
biomarkers of inflammation. Certain levels of cytokines are needed to stimulate 
an immune response in applications such as vaccines or immunotherapy where 
immune stimulation is desired. However, undesirable elevation of cytokine 
levels, as may occur in response to a drug or a device, may lead to severe side 
effects such as systemic inflammatory response syndrome or cytokine storm. 
Therefore, preclinical evaluation of a test material's propensity to cause 
cytokine secretion by healthy immune cells is an important parameter for 
establishing its safety profile. Herein, we describe in vitro methods for 
analysis of cytokines, chemokines, and type II interferon in whole blood 
cultures derived from healthy donor volunteers. First, whole blood is incubated 
with controls and tested nanomaterials for 24 h. Then, culture supernatants are 
analyzed by ELISA to detect IL-1β, TNFα, IL-8, and IFNγ. The culture 
supernatants can also be analyzed for the presence of other biomarkers secreted 
by the immune cells. Such testing would require additional assays not covered in 
this chapter and/or optimization of the test procedure to include relevant 
positive controls and/or cell types.