The Assessment of Circadian Rhythms Within the Immune System

In recent years, circadian rhythms have been observed in many aspects of the immune system, both for the innate immunity (the first line of defense against pathogens) and the adaptive immunity (a more specific set of responses, which lead to immune memory). Here, to illustrate principles to be taken into account when working on circadian rhythms in immunology experiments, two protocols will be presented. The first one aims to analyze immune parameters in blood sampled from human subjects at different times over the day: counts of different cell types among the peripheral blood mononuclear cells and cytokine secretion by monocytes and T cells after ex vivo stimulation. The second protocol describes how to follow the response of CD8⁺ T cells after immunization of mice with antigen presenting cells loaded with a peptide antigen. These two protocols are optimized for circadian experiments, and outcome measures are mainly based on flow cytometry, which allows analysis of different parameters in the same cells.