SHORT RESEARCH ARTICLE
Flow Cytometry Measurement of Reactive Oxygen Species Generation: Some Caveats and Their Solution
Mutaz Dana and Eitan Fibach
The Hematology Branch, Hebrew University–Hadassah Medical Center, Jerusalem, Israel
Correspondence: email@example.com (E.F.)
Dana M and Fibach E. Reactive Oxygen Species 10(28):171–175, 2020; ©2020 Cell Med Press
(Received: February 2, 2020; Revised: April 1, 2020; Accepted: April 2, 2020)
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.
KEYWORDS | Flow cytometry; Reactive oxygen species; Red blood cells; Thalassemia
ABBREVIATIONS | DCFDA, 2′7′-dichlorodihydrofluorescin diacetate; MCF, mean fluorescence; RBCs, red blood cells; ROS, reactive oxygen species
- Materials and Methods
- Results and Discussion
The redox state plays an important role in the normal physiological functioning (e.g., signal transduction) of the body as well as in its malfunctioning during diseases [1, 2]. The redox state depends on the balance between the oxidants, such as reactive oxygen species (ROS), and antioxidants (such as glutathione) in the cells. When the balance is tilted towards the oxidants, oxidative stress is generated. Excess ROS interact with various cellular components such as proteins, lipids and DNA, leading to cell death and organ damage .
Measurement of redox state parameters in cells and body fluids such as the blood plasma can be accomplished by various methods , and yet it is not a common practice in the clinic. A major limitation is the inadequacy of most of these methods for routine clinical laboratory use. We have measured these parameters by flow cytometry in blood cells [5, 6]. Flow cytometry is a common methodology in the clinical setting, and blood cells can be easily obtained . Because they constantly circulate, blood cells, particularly red blood cells (RBCs), can serve as biomarkers of the oxidative status of the whole body, and, thus, “report” oxidative stress. The probe 2′7′-dichlorodihydrofluorescindiacetate (DCFDA) is commonly applied for quantification of ROS . Following incubation with cells, this nonfluorescent compound is taken up and undergoes esterification by cellular enzymes into 2′7′-dichlorodihydro-fluorescin (DCFH), that get trapped inside the cells. ROS (mainly peroxides) oxidize it to the fluorescent compound 2′7′-dichlorodihydrofluorescein (DCF)  (Figure 1A). The fluorescence emitted by the cells, measured by flow cytometry, is proportional to their ROS content. Using differences between cells, such as size, granularity and/or expression of specific surface markers, permit simultaneous, multiparameter measuring of ROS in various cell types .
FIGURE 1. ROS measurement in normal and thalassemia RBCs. (A) ROS-induced fluorescence of DCF. (B and C) Normal and thalassemia RBCs were labeled with DCFDA, washed, then re-incubated in DCFDA-free PBS, and analyzed at different time points. (B) Dot-plot distributions with respect to cellular DCF fluorescence/forward light-scatter at times 0 and 60 min of normal and thalassemia RBCs. The mean fluorescence channels (MFC) are indicated;(C) The rate of change in DCF fluorescence of normal (□, n =20) and thalassemia (■, n =20) RBCs.
The extent of the cellular DCF fluorescence, which is in the core of this technique, depends, in addition to the level of ROS, on the intracellular content of DCF and the presence of intracellular quenching compounds, such as hemoglobin (Hb) in RBCs. The former depends on the amount of DCFDA added, the number of cells, the composition of the medium, and the conditions (e.g., the temperature) of incubation. These parameters can be experimentally controlled. It also depends on the DCFDA uptake by the cells and its esterification to DCFH that in turn depend on the cell characteristics such as size and membrane properties that may differ in cells of various types, stages of differentiation or senescence, or under different conditions (pathological vs. normal). Since these parameters cannot be controlled by the experimenter, a one-point measurement, which does not take into account these differences, is inadequate. A recent searching of the PubMed produced a huge number of citations for the use of DCFDA as a probe for measuring ROS, and most of these studies used one-point measurement without referring to the caveats involved.
Herein, we describe a protocol that determines the cellular rate of ROS generation by measuring the change in the DCFfluorescence with time (kinetics). In this two-phase procedure, cells are first pulsed with DCFDA, washed, and then incubated in DCFDA-free medium. The cellular ROS level was measured at different time points. For illustration, we present results with RBCs of thalassemia patients, having a high content of ROS [2, 10], compared with normal RBCs.
The results suggest that this pulse-chase procedure may be valuable for determining the influence of internal and external factors on the rate of ROS generation in various cells under different conditions.
2. MATERIALS AND METHODS
Fresh peripheral blood samples, obtained in ethylenediaminetetraacetate (EDTA)-containing tubes, from normal donors and thalassemia patients were used. The samples were obtained from the counting vials after all diagnostic laboratory tests were completed. The research was approved by the Hadassah–Hebrew University Medical Center Human Experimentation Review Board. Informed consent was obtained in all cases. The patients’ mutations and some relevant clinical parameters (transfusion and splenectomy) were previously reported . In multiply transfused patients, blood samples were obtained before transfusion, that is, at least 3 weeks after the previous transfusion.
RBCs were collected (after removing the platelet-rich plasma and the white blood cell-containing fraction), washed and diluted in phosphate-buffered saline (PBS) to 1 × 106/ml. Then, they were labeled with 0.1 mM DCFDA (Sigma) for 15 min at 37oC in the dark, as previously described [5, 6], washed twice, and further incubated in DCFDA-free PBS at 37oC. Aliquots were analyzed at time 0 of the chase incubation and at different time points thereafter.
Cellular fluorescence was measured by a fluorescence-activated cell sorter (FACS-Calibur) and the CellQueste software (Becton-Dickinson, Immunofluorometry Systems, Mountain View, CA, USA) as previously described [5, 6]. RBCs were “gated” according to their forward and side light-scatter. Their mean green fluorescence channel (MFC) was calculated. Unstained RBCs served as control.
For statistical analysis, comparisons between groups were performed using Student’s t-test and p < 0.05 was considered as significant.
3. RESULTS AND DISCUSSION
RBCs were pulsed and chased with DCFDA. Figure 1B shows the dot-plot distributions with respect to DCF fluorescence/forward light-scatter of representative samples of normal and thalassemia RBCs at time 0 and 60 min of the chase incubation. The results show a higher fluorescence in thalassemia RBCs vs. normal RBCs. The average MFC was 40 ± 15 vs. 11 ± 7 at time 0 and 70 ± 15 vs. 321 ± 25 after 60 min in thalassemia (n = 20) and normal (n = 20) RBC samples, respectively. Figure 1C shows the increase in the fluorescence with the time of the chase. It was linear during the first 1-h of incubation in both RBC types. The average rate of the increase (kinetics) in fluorescence was 4.5-fold higher in thalassemia RBCs (272/h) compared with normal RBCs (56/h) (p < 0.001).
Measurement of the DCF fluorescence in intact cells by spectrofluorometry  or flow cytometry  has been applied to determine ROS, and thereby, the oxidative status. This has been utilized mainly for research purposes, but not in the routine clinical laboratory. Flow cytometry is commonly used for clinical purposes, such as immunophenotyping of blood and bone marrow samples in malignancy and immunodeficiency. The results are usually expressed as a percent of positive cells, but the extent of positivity of the population—in arbitrary units of mean fluorescence channel rather than absolute quantitative values. It is, however, suitable for comparison purposes such as detecting differences between normal and abnormal cells. The cells’ DCF fluorescence following labeling with DCFDA is proportional to their ROS content. However, in addition, as discussed by Loetchutinat et al. using spectrofluorometry , it depends on the cellular concentration of DCFH that, among other factors, depends on the rate of DCFDA uptake and esterification (Figure 1A). Various cells may differ in these activities; thus, relying on one time-point measurement may be inappropriate for quantification of the cellular ROS. This problem may be shared by other probes that rely on esterase activity, such as calcein-AM and Fluo-4 for measuring intracellular free iron  and calcium , respectively.
To overcome this caveat, we measured the rate of change (the kinetics) in the cellular fluorescence of DCF-preloaded cells. The cells are first loaded with DCFDA and then washed to remove external DCFDA and incubated (chased) in DCFDA-free PBS. Any increase in the cellular fluorescence during the chase is the result of a shift of the non-fluorescence DCFH to the fluorescence DCF due to continued ROS generation. The results showed that although there was a significant difference in fluorescence between thalassemia and normal RBCs (Figure 1B), the increases in fluorescence with time (the kinetics) were linear in both cell types during the 1-h chase regardless the basal fluorescence at time 0.
The cellular fluorescence may be affected by intracellular compounds that may quench the fluorescence, most notably Hb in RBCs. Thalassemia RBCs are smaller in size (MCV) and contain less Hb content (MCH) and concentration (MCHC) than normal RBCs . These differences may affect the basal fluorescence but, since they are constant during the chase, they do not change the kinetics of fluorescence (i.e., ROS generation). Measuring the RBC indexes (MCV, MCH, and MCHC) by an electronic blood counter showed the expected difference between the average values of the thalassemia and the normal samples (p < 0.005), but demonstrated no change within each sample during the 1-h chase incubation (p = 0.2). In addition, spectrofluorometric analysis of the extracellular medium (using the benzidine method ) indicated no loss of Hb due to hemolysis during the chase.
The cellular ROS generation may be affected also by external conditions including external sources of ROS, such as peroxides generated by polymorphonuclear leukocytes (PMNs) and monocytes, or antioxidantsandantioxidantenzymespresentinthe serum. In the experiments described above, no such sources were present as the RBCs tested were isolated and the chase was carried out in serum-free PBS. Of course, the addition of oxidants (e.g., hydrogen peroxide) or antioxidants (e.g., catalase), metabolites (e.g., glucose) or anti-metabolites as well as changing the temperature of incubation may affect the rate of ROS.
In conclusion, this report introduces a novel protocol for comparing the rate of ROS generation, avoiding certain caveats due to differences between cells. We propose that this protocol may be valuable for determining the influence of internal and external factors on the rate of ROS generation by different types of cells.
CONFLICTS OF INTEREST STATEMENT
The authors declare no conflict of interest.
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