Fluoroskan ascent fluorescence spectrophotometer

The permeability of the cell envelope was examined by measuring the fluorescence intensity of the cells labeled by fluorescein diacetate (FDA, Aladdin Reagents (Shanghai) Co., Ltd., Shanghai, China) according to previous procedures [38 (link)]. Cell suspensions (cell density reached 10⁶ cells/mL) of 4.0 mL was mixed with 0.5 mL FDA acetone solution (2 mg/mL) and vibrated at 32 °C for 5 min before detection with a Fluoroskan Ascent Fluorescence Spectrophotometer (Thermo Labsystems Inc., PA, USA). The maximal excitation wavelength for FDA was 485 nm and the emission wavelength was 538 nm.

The antioxidant activity of extracts was determined by using a PSC assay developed in our lab [24] (link) with modifications [25] . Just prior to use in the reaction, 107 µL of 2.48 mM dichlorofluorescein diacetate was hydrolyzed to dichlorofluorescein with 893 µL of 1.0 mM KOH for 5 min in a vial to remove the diacetate moiety and then diluted with 7 mL of 75 mM phosphate buffer (pH 7.4). ABAP (200 mM) was prepared fresh in the buffer and was kept at 4°C between runs. In an assay, 100 µL of extracts was diluted in 75 mM phosphate buffer (pH 7.4) and then transferred into reaction cells on a 96-well plate, and 100 µL of dichlorofluorescein was added. The 96-well plate was loaded into a Fluoroskan Ascent fluorescence spectrophotometer (Thermo Labsystems, Franklin, MA), and the solution in each cell was mixed by shaking at 1200 rpm for 20 s. The reaction was then initiated by adding 50 µL of ABAP from the autodispenser on the equipment. Each set of dilutions for a replicate and control was analyzed three times in adjacent columns. The reaction was carried out at 37°C, and fluorescence was monitored at 485 nm excitation and 538 nm emission with the fluorescence spectrophotometer. The buffer was used for control reactions. Data were acquired with Ascent software, version 2.6 (Thermo Labsystems). Data were reported as mean ± SD with three replicates.