Immunophenotyping is proving crucial to understanding the role of the immune system in health and disease.High-through-put flow cytometry has been used extensively to reveal changes in immune cell composition and func...Immunophenotyping is proving crucial to understanding the role of the immune system in health and disease.High-through-put flow cytometry has been used extensively to reveal changes in immune cell composition and function at the single-cell level.Here,we describe six optimized 11-color flow cytometry panels for deep immunophenotyping of human whole blood.A total of 51 surface antibodies,which are readily available and validated,were selected to identify the key immune cell populations and evaluate their functional state in a single assay.The gating strategies for effective flow cytometry data analysis are included in the protocol.To ensure data reproducibility,we provide detailed procedures in three parts,including(1)instrument characterization and detector gain optimization,(2)antibody titration and sample staining,and(3)data acquisition and quality checks.This standardized approach has been applied to a variety of donors for a better understanding of the complexity of the human immune system.展开更多
The immune system defends the body from infection and plays a vital role in a wide range of health conditions.Metabolism afects a series of physiological processes,including those linked to the function of human immun...The immune system defends the body from infection and plays a vital role in a wide range of health conditions.Metabolism afects a series of physiological processes,including those linked to the function of human immune system.Cellular metabolism modulates immune cell activation and cytokine production.Understanding the relationship between metabolism and immune response has important implications for the development of immune-based therapeutics.However,the deployment of large-scale functional assays to investigate the metabolic regulation of immune response has been limited by the lack of standardized procedures.Here,we present a protocol for the analysis of immune response using standardized whole-blood stimulation with metabolism modulation.Diverse immune stimuli including pattern recognition receptor(PRR)ligands and microbial stimuli were incubated with fresh human whole blood.The metabolic inhibitors were used to modulate metabolic status in the immune cells.The variable immune responses after metabolic interventions were evaluated.We described in detail the main steps involved in the whole-blood stimulation and cytokines quantifcation,namely,collection and treatment of whole blood,preparation of samples and controls,cytokines detection,and stimulation with metabolic interventions.The metabolic inhibitors for anabolic pathways and catabolic pathways exert selective efects on the production of cytokines from immune cells.In addition to a robust and accurate assessment of immune response in cohort studies,the standardized whole-blood stimulation with metabolic regulation might provide new insights for modulating immunity.展开更多
基金supported by the National Key Research and Development Program of China(2021YFA1301000)Shanghai Municipal Science and Technology Major Project(Grant No.2017SHZDZX01).
文摘Immunophenotyping is proving crucial to understanding the role of the immune system in health and disease.High-through-put flow cytometry has been used extensively to reveal changes in immune cell composition and function at the single-cell level.Here,we describe six optimized 11-color flow cytometry panels for deep immunophenotyping of human whole blood.A total of 51 surface antibodies,which are readily available and validated,were selected to identify the key immune cell populations and evaluate their functional state in a single assay.The gating strategies for effective flow cytometry data analysis are included in the protocol.To ensure data reproducibility,we provide detailed procedures in three parts,including(1)instrument characterization and detector gain optimization,(2)antibody titration and sample staining,and(3)data acquisition and quality checks.This standardized approach has been applied to a variety of donors for a better understanding of the complexity of the human immune system.
基金National Key Research and Development Program of China(2021YFA1301000)Shanghai Municipal Technology Standards Project(23DZ2202600)Shanghai Municipal Science and Technology Major Project(Grant No.2017SHZDZX01).
文摘The immune system defends the body from infection and plays a vital role in a wide range of health conditions.Metabolism afects a series of physiological processes,including those linked to the function of human immune system.Cellular metabolism modulates immune cell activation and cytokine production.Understanding the relationship between metabolism and immune response has important implications for the development of immune-based therapeutics.However,the deployment of large-scale functional assays to investigate the metabolic regulation of immune response has been limited by the lack of standardized procedures.Here,we present a protocol for the analysis of immune response using standardized whole-blood stimulation with metabolism modulation.Diverse immune stimuli including pattern recognition receptor(PRR)ligands and microbial stimuli were incubated with fresh human whole blood.The metabolic inhibitors were used to modulate metabolic status in the immune cells.The variable immune responses after metabolic interventions were evaluated.We described in detail the main steps involved in the whole-blood stimulation and cytokines quantifcation,namely,collection and treatment of whole blood,preparation of samples and controls,cytokines detection,and stimulation with metabolic interventions.The metabolic inhibitors for anabolic pathways and catabolic pathways exert selective efects on the production of cytokines from immune cells.In addition to a robust and accurate assessment of immune response in cohort studies,the standardized whole-blood stimulation with metabolic regulation might provide new insights for modulating immunity.
