Flow cytometry has become a basic of biological research and clinical diagnostics, and its application has been crucial to numerous advances in immunology and cell biology. However, several issues remain when considering intracellular stainings, especially in the context of a daily routine use and in multicenter clinical research protocols including large cohorts of patients. The requirements for multiple protocol steps are not only time-consuming but also frequently associated with high cell loss and nonspecific binding or reduced fluorescence. These drawbacks make standardized intracellular flow cytometry use in multicenter studies struggling. As a consequence, intracellular flow cytometry has mostly remained a tool for experimental and clinical research. In the current chapter, we will complete flow cytometry protocols described in the previous edition by presenting novel intracellular protocols usable in clinic. These present with many advantages including shorter time-to-results, one-step whole blood procedures, lyse-no-wash-no-centrifuge protocols, improved staining quality, and lyophilized coated reagents in ready-to-use tubes. This opens novel perspectives for standardization and feasibility in clinical studies, for drug efficacy monitoring and for patients' stratification within a context of personalized medicine. Here, we present illustrative examples taken from septic patients' immunomonitoring. We consider the evaluation of myeloperoxidase and lactoferrin expressions in neutrophils, FOXP3 lymphocyte expression, and STAT5 phosphorylation in lymphocyte subsets.
Keywords: FOXP3; Intracellular flow cytometry; Lactoferrin; Myeloperoxidase; Neutrophil; Regulatory T cells; STAT5; Sepsis.