Analysis of the Immune Response by Standardized Whole‑Blood Stimulation with Metabolism Modulation

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.

Deep Immunophenotyping of Human Whole Blood by Standardized Multi‑parametric Flow Cytometry Analyses

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.

Wheat morphological and biochemical responses to copper oxide nanoparticles in two soils

The widespread application of copper oxide nanoparticles(CuO NPs)in agricultural production has caused growing concerns about their impact on crops.In this study,wheat root elongation was used to evaluate the toxic effect concentrations of CuO NPs in two soils with differing properties,collected from farmlands in Guangdong(GD)and Shandong(SD)provinces,China.Plant morphological and biochemical properties were also assessed to explore the toxicity mechanism of CuO NPs on wheat seedlings.The root elongation results revealed lower toxic effect concentration values in the plants grown in GD soil than in SD soil.Furthermore,the treatment with CuO NPs at 200 mg Cu kg^(-1) significantly reduced wheat root and shoot biomass by 35.8%and 15.8%,respectively,in GD soil.Electron microscopy showed that CuO NPs deformed wheat roots and entered leaf cells,causing deformation and damaging the cell structure.The CuO NP treatments also decreased chlorophyll content,increased antioxidant enzyme activity,and increased membrane lipid peroxidation in wheat leaves.The addition of CuO NPs significantly reduced the Zn(by 17.3%)and Fe(by 26.9%)contents in the leaves of plants grown in GD and SD soils,respectively.However,the contents of Cu,Mg,and Mn were increased by 27.4%–52.5%in GD soil and by 17.9%–71.6%in SD soil.These results suggested that CuO NPs showed greater toxicity to wheat plants grown in acidic soil than in alkaline soil and that the adverse effects of CuO NP treatments on wheat seedlings were due to a combination of CuO NPs and released Cu^(2+).