Dcfh da

2, 7 -Dichlorofluorescin diacetate (DCFH-DA, Sigma), a stable non-fluorescent compound, was produced from dissolving 0.02 mmol of DCFH-DA in 1 mL of DMSO. DCFHDA underwent deacetylation by intracellular esterases when crossing the membrane and converted to DCFH. In the presence of active radicals within the cell, the non-fluorescent DCFH can oxidize and produce the highly fluorescent form DCF22. The cells were harvested and washed within PBS and incubated with 50 μmol/L 2, 7 -Dichlorofluorescin diacetate (DCFH-DA, Sigma) at 37 °C for 2 h. Then the cells were washed and placed on ice in the dark. Before reading, 5 μl of Propidium Iodide (PI; Sigma-Aldrich, MO, USA) was added to the samples. The analysis was performed with the FACS Calibur FCM. The labeled cells were excited at 488 nm, and the emissions were detected at 520 nm and 630 nm.

Experiments were performed according to the instructions of the kit, and levels of ROS and iNOS in erythrocytes were measured with the fluorescent probe 2’-7’-dichlorodihydrofluorescein-diacetate (DCFH-DA, Sigma, USA), which penetrates cells and hydrolyzes to non-fluorescent dichlorodihydrofluorescein (DCFH). NO production was measured by the fluorescence probe DAF-FM (Sigma, USA). Briefly, purified erythrocytes (1 × 10⁵ cells/ml) were incubated with S. aureus (10⁶ CFU/ml), A. hydrophila (10⁶ CFU/ml), and E. coli (10⁶ CFU/ml) at 1 h, 2 h, and 4 h, and suspensions were collected at these time points. The mixtures were incubated with 1 μmol/ml DCFH-DA (Sigma, USA) or DAF-FM (Sigma, USA) for 25 min at 37°C in a sheltered room, and then, the cells were washed three times with 0.9% sterile buffered saline and resuspended in 500 μL 0.9% sterile buffered saline for analysis with a FACScan flow cytometer (BD Biosciences, USA).

TNF-α in the supernatant of the RAW264.7 cells treated by β
₁-AA was quantified using the TNF-α ELISA kit (Boster, Pleasanton, USA) according to the manufacturer’s protocol. The intracellular ROS level in macrophages was quantified by using the 2,7-dichlorofluorescein diacetate (DCFH-DA; Sigma, St Louis, USA). The macrophages were seeded in 6-well plates at 1.6×10
⁵ cells/well and allowed to adhere overnight. Subsequently, the medium was replaced by fresh medium with different β
₁-AA concentrations (10
^–6 M, 10
^–7 M, and 10
^–8 M), negative IgG (10
^–7 M), metoprolol (3× 10
^–7 M), LPS (10 ng/mL; Sigma-Aldrich) or fresh medium alone and incubated for 24 h. The plates were washed twice with PBS and incubated with DCFH-DA (10 μM) at 37°C for 20 min. The mean fluorescence intensity (MFI) of the cells was determined at the best excitation wavelength of 485 nm and emission wavelength of 525 nm using the microplate reader.