Flow cytometry uses light scattering, light excitation and emission of fluorochrome molecules to gather detailed information about specific cells or particles of interest. Using antibody-fluorophore conjugates flow cytometry allows researchers to draw phenotype-dependent conclusions about cellular phenotype and function. The fluorophores present on bound antibodies excited by lasers yield detailed immune response data that make flow cytometry a vital tool for quantifying the effectiveness of vaccine candidates.
A successful vaccine antigen will stimulate an adaptive immune response which will result in the immune system retaining a “memory” of the antigen. This in turn allows for a more efficient response during subsequent exposures. The cascade mechanism that allows the immune system to mount an appropriate response against invading cells (as well as rogue self-cells) is complex. Flow cytometry allows for highly customizable assays for details monitoring of specific response. Lymphocytes isolated from test animal treated with trial antigen can be exposed to peptides from the same antigen can be stained for surface markers and intracellular cytokines to determine their phenotypes and functional purposes.
If a vaccine is successful in prompting a response, an increase in intracellular cytokines (TNFα, IFNγ, IL-2, etc.) would be observed in T-lymphocyte populations exposed to the antigen peptides (Fig. 1). This indicates that the antigen in question has successfully provoked an adaptive immune response, and future exposures to the same antigen will elicit the same type of response.