Cellular redox state determinants are traditionally studied using fluorescent microscopy and immunoblot analysis; however, no procedure has been devel-oped for simultaneous measurement in various immune cell subsets. Here, we present a flow cytometry assay for measuring antioxidant defense systems and reactive oxygen species simultaneously in T, B, and natural killer lympho-cytes. We describe steps for preparing and treating peripheral blood mononu-clear cells, surface and dye staining, cell fixation/permeabilization, and intra-cellular staining. We then detail machine standardization, acquisition, and analysis.
Pitsillidou, C., Muradore, I., Pontarini, E., Bertolotti, M., Roberto, A. (2023). A redox-based characterization of human immune cell subsets by polychromatic flow cytometry. STAR PROTOCOLS, 4(4), 1-17 [10.1016/j.xpro.2023.102632].
A redox-based characterization of human immune cell subsets by polychromatic flow cytometry
Pitsillidou, C;
2023
Abstract
Cellular redox state determinants are traditionally studied using fluorescent microscopy and immunoblot analysis; however, no procedure has been devel-oped for simultaneous measurement in various immune cell subsets. Here, we present a flow cytometry assay for measuring antioxidant defense systems and reactive oxygen species simultaneously in T, B, and natural killer lympho-cytes. We describe steps for preparing and treating peripheral blood mononu-clear cells, surface and dye staining, cell fixation/permeabilization, and intra-cellular staining. We then detail machine standardization, acquisition, and analysis.
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