Immunophenotypic Assessment of Natural Killer Cell Degranulation and Monocyte Function from Cryopreserved Peripheral...

Natural killer (NK) cells and monocytes are central components of the innate immune system, playing critical roles in host defense, immune surveillance, and inflammation. Functional alterations in these cell populations are implicated in cancer, infectious diseases, cardiovascular disease, and responses to environmental and psychosocial stressors. Accurate measurement of NK cell and monocyte distribution and function is therefore essential for both basic and translational immunology. In large-scale or longitudinal studies, cryopreserved peripheral blood mononuclear cells (PBMCs) are commonly used to enable standardized analyses across time and sites; however, cryopreservation and thawing can adversely affect cell viability and function. This chapter presents a reproducible protocol for the cryopreservation, thawing, and functional assessment of NK cells and monocytes from human PBMCs. PBMCs are cryopreserved using a 9:1 ratio of fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO), allowing long-term storage at ultra-low temperatures while preserving cellular integrity. The thawing process utilizes a nuclease to minimize cell clumping, enhancing cell recovery and viability. Following thawing, PBMCs are prepared for downstream functional assays. NK cell function is assessed using a standard degranulation assay following coculture with K562 target cells, providing a robust measure of cytotoxic capacity. Together, this protocol offers a practical guide for the reliable measurement of NK cell and monocyte distribution and function from cryopreserved PBMCs, supporting high-quality immunological analyses in clinical and population-based research.