OMIP ‐038: Innate immune assessment with a 14 color flow cytometry panel

PROFILING the innate immune system of young children in a state of health and disease, is an important but arduous task, plagued with restrictions and limitations.Once blood samples are obtained, no matter how limited in volume, they are extremely precious.Therefore great care and consideration must be taken in how these samples are assayed.The purpose of the panel presented here is to assess the innate immune response in whole blood after Toll-like receptor (TLR) ligand or cytokine stimulation, at the single-cell level.In addition to the identification of many of the innate and innateadaptive interface immune cell subsets, this panel also enables the detection of key intracellular cytokines involved in the immune response.This panel was optimized for the evaluation of TLR-ligand and cytokine stimulated whole blood, which has been fixed and frozen, prior to flow cytometric analysis to allow batched analysis to reduce technical artifacts.The panel presented here is an expansion of a panel previously described in Refs.(1-4), but expanded here to capture most of the major innate immune subsets found in peripheral blood and their key functional cytokines.Although the specific application of this panel was to identify the baseline difference between groups of healthy children, shown in Ref. (5), this panel is readily applied to a wide range of normal and diseased-states in humans. BACKGROUNDThe innate immune system instructs the adaptive immune response.Thus, an understanding of the innate immune response is essential in vaccine development and prediction of protective host immune responses following infection.Often human blood samples are limited in volume; this is especially true for pediatric samples.These precious samples require an efficient multi-color analysis to extract the maximum information out of the smallest of samples.The focus of this panel is to precisely characterize the innate immune responses on the single cell level in a highly standardized way, allowing for the identification of specific cell subtypes, the exclusion of potential contaminating cells, and the incorporation of innateadaptive interface cells and their function.Our immune profiling approach allows for exactly that; by the determining of cell phenotype and frequencies, differentiating between various cell subtypes, and quantifying functional cytokine response (Table 1).