OPTIMIZATION OF A 6 COLOR INTRACELLULAR CYTOKINE STAINING PROTOCOL FOR USE IN RHESUS MACAQUES IMMUNIZED WITH MALARIA VACCINE CANDIDATES

Abstract

Intracellular Cytokine Staining (ICS) is a method used to quantitatively determine the phenotype of cells and their ability to produce cytokines when stimulated with antigen. As part of a larger study to examine the safety and immunogenicity of a malaria vaccine candidate, we have optimized an ICS assay to detect cytokine production in antigen stimulated immune cells of rhesus macaques. We have seen that the addition of IL-7 and IL-15 to culture during stimulation can increase antigen-specific cytokine production in both CD4+ and CD8+ T cells without increasing background levels beyond which signal can no longer be detected. Background read-outs of cytokine were greatly reduced by including a CD3 gate and intentionally choosing fluorochrome conjugated antibodies to avoid spectral overlap and data spread, allowing better detection of antigen-specific responses.