Life launch attune nxt flow cytometer

The cells were irradiated with 8 mJ/cm² UVB, and then the cells were washed once with 1 mL PBS, and a culture solution containing AYC-CS-E (0.125 to 64 µL/mL) was added. After 24 h, cells were harvested and stained with 5 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich) at 37 °C for 30 min and washed twice with PBS. The intracellular ROS expression levels (DCFH-DA-positive cells) of each cell were analyzed with a Life Launch Attune Nxt Flow Cytometer (ThermoFisher Scientific, Waltham, MA, USA) using FlowJo software (Version 10; Tree Star, Inc., Ashland, OR, USA).

Intracellular ROS intensity was measured by Reactive Oxygen Species Assay Kit (Beyotime, Shanghai) as described in the manufacturer's instructions. DCFH-DA fluorescence was measured by using Life Launch Attune NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). Images were taken using Nikon Eclipse TI fluorescence microscope (Nikon Corporation, Tokyo). Data were analyzed using FlowJo software (version 10).

To analyze the viability of immature and mature DCs, DCs (0.5 × 10⁶ cells per well in 500 μL complete RPMI1640 media) were incubated with or without 100 ng/mL lipopolysaccharide (LPS; Invitrogen, San Diego, CA, USA) as a positive control for DC maturation for 2 h, and then treated with various concentrations (1–50 μg/mL) of AYC-EVs for 24 h. After stimulation, cell viability was analyzed using the EZ-Cytox Cell Viability Assay Kit (DoGen, Seoul, Korea), according to the manufacturer’s protocol. Briefly, 50 μL of EZ-Cytox kit reagent was added to each well and incubated at 37 °C for 60 min. After incubation, absorbance at 450 nm was measured using a microplate reader (Molecular Devices Inc., San Jose, CA, USA). For cytotoxicity analysis, cells (non-, LPS-, AYC-EV-, AYC-EV, and LPS-treated DCs) were harvested 24 h after AYC-EV treatment and stained with Annexin V and propidium iodide (PI) using FITC Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. Annexin V/PI-stained cells were analyzed using a Life Launch Attune Nxt Flow Cytometer (Thermo Fisher Scientific) and FlowJo software (Version 10; Tree Star, Inc., Ashland, OR, USA).