Cold staining buffer

Lymphocyte apoptosis was identified by flow cytometry analyses using an APC Annexin-V apoptosis detection kit (BioLegend, catalog no. 640930). First, the cells were stained for monoclonal antibodies. A cold staining buffer (BioLegend, catalog no. 420201) was used to wash the cells. The cells were then resuspended in an annexin-V binding buffer (BioLegend, catalog no. 422201). A total of 5 μL annexin-V (8 µg/mL) was added to each tube for fifteen minutes in the dark at normal temperatures. The annexin-V binding buffer was then added. The analysis of the cells was carried out utilizing FACSDiva software (BD Biosciences, San Diego, CA, USA). Data were shown as percentages of annexin-V⁺ lymphocytes. The positive threshold was constructed using non-stained controls.

HUVEC cells were seeded in 24-well plates at a density 50,000 cells per well and cultured for 24 h under standard conditions (volume 0.3 mL). Next, the cells were exposed to the tested compounds (2, 3, 4, 6 or 10) at the concentrations of 0.1 and 0.3 µmol/mL or ascorbic acid as reference substance. After 24 h treatment, control and treated cells were collected (washed with PBS, and harvested with accutase), and transferred to Eppendorf tubes. The cells were centrifuged (5 min, 220× g). Then, the supernatant was discarded, cold staining buffer (Biolegend, London, UK) was added (0.5 mL), and the cells were centrifuged (200× g, 5 min.). The solution was discarded, and the cells were resuspended in 200 µL of cell staining buffer. Then, 2 µL of H₂DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) (final concentration 2 µmol/L) was added, mixed and incubated for 30 min in the dark at room temperature. The final volume of sample was 250 µL. The fluorescence was measured by flow cytometry (CytoFlex, blue laser, 480 nm, Beckman-Coulter, Indianapolis, IN, US). The results were analyzed using Kaluza 2.1 (Beckman-Coulter Inc., Brea, CA, USA) software. In total, 10,000 cells were analyzed from each sample and the data were presented as the mean ± SD of three independent experiments. The coefficients of variation for the assay was determined (CV = 8.26%, n = 3).

MCF-7 and MDA-MB-231 cells were cultured according to the procedure described in Section 3.6. After 24-h incubation, the cells were exposed to the tested compounds at concentrations equaling ½ of IC₅₀ value of each compound or metformin (500 µmol/L) as reference substance. 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH; (Sigma Aldrich, St. Louis, MO, USA) was used as positive control. After 24 h treatment, control and treated cells were collected to Eppendorf tubes, and centrifuged (5 min, 1100 rpm). The cells were washed with cold staining buffer (Biolegend, London, UK) followed by 30 min incubation with 2 µL of H₂DCFDA (2′,7′-dichlorodihydrofluorescein diacetate; Thermo Fisher Scientific, Waltham, MA, USA) (final concentration 2 µmol/L). The fluorescence of the samples was measured by flow cytometry (CytoFlex, blue laser, 480 nm, Beckman-Coulter, Indianapolis, IN, USA), and the results were analyzed using Kaluza 2.1 (Beckman-Coulter, Indianapolis, IN, USA) software. 10,000 cells were analyzed from each sample (plate well) and the data are presented as the mean ± SD, n = 3. The coefficient of variation for the assay was determined (CV = 8.68%, n = 3).