Cellquest program

Cultured MDA-MB-231 cells were treated grown to a density of 1x10⁵ cells and treated with different concentrations of metformin for 24 h. After treatment, cells were centrifuged at 500Xg for 5 minutes and the pellet was washed with 1X PBS. Cells in the pellet were then suspended in 1X Annexin binding buffer and stained with fluorescein isothiocyanate [FITC]-conjugated annexin V and propidium iodide [PI] for 30 min at room temperature in the dark following the manufacturer’s protocol (TACS Annexin V-FITC Apoptosis Detection Kit, R&D Systems, 4830-01-K) [20 (link)]. After incubation cells were analyzed for apoptotic and necrotic population using BD FACSCalibur (BD Biosciences). The normal healthy cells, early apoptosis, late apoptosis and necrotic populations were represented by annexin V-negative/PI-negative population, annexin V-positive/PI-negative, annexin V-positive/PI-positive and annexin-negative/PI-positive cells, respectively. The data were analysed using Cell Quest program from Becton-Dickinson [21 (link)].

For analysis of T cell profiles, PBMCs cultured for 48 h were resuspended (5 × 10⁵ cells/mL) in Hank's medium (Sigma, St. Louis, MO, USA), washed three times (400 ×g, 4°C, 10 min), and incubated in Hank's medium supplemented with 10% inactivated human AB+ serum. Subsequently, the samples were labeled with corresponding surface antibodies for each T cell profile. The cells were washed to remove excess antibody and then permeabilized and fixed with the addition of 500 μL Cytoperm/Cytofix (BD Biosciences, San Jose, CA, USA). Next, the samples were labeled with corresponding intracellular antibodies for each T cell profile and washed again to remove excess antibodies. Finally, the cells were resuspended in 500 μL PBS containing 0.5% paraformaldehyde and stored at 4°C in the dark until flow cytometry analysis. The samples were labeled with antibodies (BD Biosciences, San Jose, CA, USA) for T cell profiles, including Th1 (IFN-γ-FITC, T-bet-PE, and CD4-PerCP), Th2 (IL-4-FITC, GATA-3-PE, and CD4-PerCP), Th17 (IL-17-FITC, RORγT-PE, and CD4-PerCP), and Treg (CD25-FITC, FoxP3-PE, and CD4-PerCP). A FACSCalibur cytometer (Becton-Dickinson, Mountain View, CA, USA) was used for the acquisition of events (100,000 events/tube) and the data were analyzed using the CellQuest program (Becton-Dickinson).

Cells were seeded in 12-well plates (1x10⁵ cells/well) in complete medium and treated with terpinen-4-ol at several concentrations for 72 h. Annexin V was detected according to the manufacturer’s protocol. The cells were washed with PBS and then incubated in a solution of the membrane-impermeable nuclear dye propidium iodide. The cells were then immediately analyzed by flow cytometry [FACSCalibur (Becton Dickinson, CA)], and the results were analyzed with the CELLQuest program (Becton Dickinson).

Cell cycle analysis was performed by propidium iodide (PI) staining. HCT116 or HT29 cells were treated with Ursolic acid for 24 h and collected and fixed in 70% ethanol. The cells were then incubated at 37 °C with 0.1% ribonuclease A in PBS for 30 min and suspended in PBS containing 30 μg/mL PI for 30 min at room temperature. Sub-G1 accumulation was evaluated from the stained cells by FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA) using the Cell Quest program (Becton Dickinson, Franklin Lakes, NJ, USA).

Flow cytometry was the method of choice to study the expression of several surface activating or inhibitory receptors on patients stimulated but quiescent cells. For activated T lymphocyte and NK cell evaluation we analysed CD3-FITC, CD16-Cy5, CD54-PE, CD56-PE, Siglec7-PE, CD226-PE (DNAM-1), CD11a, CD50 (ICAM-3), NKG2A (CD159a), 2B4 (CD244), NKG2D (CD314) and NKp46 (CD335) (S1 Table). Stained cells (20,000) were collected using a FACS Scalibur cytometer (Becton-Dickinson, Mountain View, CA, USA). For white blood cells 50,000–100,000 events were collected. Data were further analysed using the CellQuest program (Becton Dickinson) and Flowing Software version 2.5.1, to obtain the mean fluorescence intensity (MFI) and the percentage of different stained populations.

Based on cell cycle method shown in Yun et al.’s paper [29 (link)], cell cycle analysis was conducted in K562 cells treated with or without FC by propidium iodide (PI) staining. Cell cycle distributions were calculated by FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA) by using the Cell Quest program (Becton Dickinson).