*2.5. Measurement of Reactive Oxygen Species (ROS) Production*

2- , 7- -Dichlorofluorescin diacetate (DCFDA) is a fluorogenic dye that measures hydroxyl, peroxyl and other ROS activities within the cell. This study used a DCFDA Cellular ROS Detection Assay Kit (ab113851, Abcam) to measure intracellular ROS according to the manufacturer's protocol. DCFDA-labeled erythrocytes were treated with different concentrations of tert-butyl hydroperoxide (TBHP) (5 mM, 10 mM, 50 mM and 100 mM) at 37 ◦C for 30 min. TBHP mimics ROS activity to oxidize DCFDA to fluorescent DCF. Finally, the mean fluorescence intensities obtained from 50,000 erythrocytes were measured using FACSCalibur (Becton Dickinson, NJ, USA). All samples were analyzed in triplicate, and the intraassay CV was 4.1 ± 0.7%.

#### *2.6. Erythrocyte Intracellular pH*

As our previous study presented [25], erythrocytes were loaded with the fluorescent pH indicator carboxy SNARF-1 (1 μM, Invitrogen) at 37 ◦C for 30 min in the dark and then washed with HBSS (Sigma) at 2500× *g* for 5 min. SNARF-1-loaded cells were incubated with pH-controlled normal K+-balanced buffer (137.9 mM NaCl, 5.33 mM KCl, 0.441 mM KH2PO4, 4.17 mM NaHCO3, 0.338 mM Na2HPO4, 5.56 mM glucose, and 20 mM HEPES, pH = 7.5) and high K+-balanced buffer at different pH values (43.7 mM NaCl, 100 mM KCl, 0.441 mM KH2PO4, 4.17 mM NaHCO3, 0.338 mM Na2HPO4, 5.56 mM glucose, and 20 mM HEPES, pH = 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0) containing 10 μM nigericin (Invitrogen). The pH was always adjusted at RT prior to use. The pH-dependent spectral shifts exhibited by SNARF-1 allowed calibration of the pH response in terms of the ratio of fluorescence intensities measured at two different wavelengths, FL2 and FL3, in a FACSCalibur (λ1 = 580 nm and λ2 = 600~640 nm and fixed excitation at 514 nm), as described in the manufacturer's protocol (Invitrogen). All samples were analyzed in triplicate, and the intraassay CV was 3.7 ± 0.9%. The equation is as follows:

$$\mathbf{p}\mathbf{H} = \mathbf{p}\mathbf{K}\_{\mathbf{a}} - \log\_{10}\left[\frac{\mathbf{R} - \mathbf{R}\_{\mathbf{B}}}{\mathbf{R}\_{\mathbf{A}} - \mathbf{R}} \times \frac{\mathbf{F}\_{\mathbf{B}(\lambda 2)}}{\mathbf{F}\_{\mathbf{A}(\lambda 2)}}\right] \tag{1}$$

pKa values: 7.5 for carboxy SNARF-1;

R: The ratio Fλ1/Fλ2 of fluorescence intensities (F) measured at λ1 and λ2; The subscript A represents the limiting values at the acidic endpoints of the titration; The subscript B represents the limiting values at the basic endpoints of the titration.
