7 aad

For the evaluation of apoptotic cells, 3 × 10⁵ CD14+ monocytes/well were cultured in RPMI 1640 (Gibco, Karlsruhe, Germany) with FBS (10%, v/v; Invitrogen, Waltham, MA, USA) and 1% (v/v) penicillin/streptomycin solution (Gibco, Karlsruhe, Germany) in a 5% CO₂ atmosphere at 37 °C. The cells were either stimulated with 100 ng/mL of TNF-α (PeproTech, Rocky Hill, NJ, USA) or 4% dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) for 24 h. The 4% DMSO induced obvious apoptosis, according to a previous study; thus, it served as the positive control group [27 (link)]. The CD14+ monocytes were washed twice with cold PBS with 1% FBS; then, we resuspended the cells in Annexin V Binding Buffer (BioLegend, San Diego, CA, USA) at a concentration of 1 × 10⁶ cells/mL. The cells were stained with Annexin V-FITC (1:50, BioLegend, San Diego, CA, USA) for 20 min at room temperature. After being washed with Annexin V Binding Buffer, the 7-AAD (Cayman, Ann Arbor, MI, USA) was also stained and incubated for 10–15 min at room temperature. The percentage of apoptotic cells was measured using flow cytometry. The apoptotic cells were defined as Annexin V-positive and 7-amino actinomycin D (AAD)-negative [28 (link)].

Annexin V (#AVK005, Strong Biotech Corporation, Taipei, Taiwan)/7AAD (#11397, Cayman, Ann Arbor, MI, USA) was used to detect apoptosis. After blue light irradiation, the cells were treated with 10 μg/mL annexin V-fluorescein isothiocyanate and 1 μg/mL 7AAD for 30 min, followed by flow cytometry using an Accuri™ C6 instrument (Becton-Dickinson, Mansfield, MA, USA).