Flow Cytometry Measurement of Reactive Oxygen Species Generation: Some Caveats and Their Solution

Abstract

The redox state is very important in normal physiology and pathology. Yet, its measurement is not practiced within the clinic. The explanation is principally methodological—the inadequacy of the methods for application in the clinical laboratory. Flow cytometry is routinely utilized in hemato-oncology and immunology. Measuring of the cellular fluorescence by flow cytometry following loading with 2'7'-dichlorodihydrofluorescin diacetate (DCFDA) has become a standard experimental method for quantification of reactive oxygen species (ROS). However, the DCF fluorescence depends on the probe uptake and esterification as well as the presence of intracellular quenchers. Cells of various types, stages of differentiation or senescence, or under different conditions (e.g., pathological vs. normal) may differ in these respects. A one-time measurement does not take into consideration these differences. Herein, we describe a protocol that overcomes these caveats by determining the change in DCF fluorescence with time as a measure of the rate of ROS generation. Normal (n = 20) and thalassemia (n = 20) red blood cells (RBCs) samples were loaded with DCFDA, washed, and then incubated in a DCFDA-free medium. DCF fluorescence was measured at different times of incubation by flow cytometry and the mean fluorescence (MFC) calculated. The results showed that the rate of increase in the DCF fluorescence was linear in both types of RBCs, regardless of their basal level and hemoglobin content, with thalassemia RBCs demonstrating a 4.5-fold higher rate (265/h) compared with normal RBCs (58/h) (p < 0.001). In conclusion, this pulse-chase procedure may be used for flow cytometry clinical evaluation of general and cell type-specific oxidative stress under normal and pathological conditions.