Facscan flow cytometer

Induction of apoptosis was assessed using a kit obtained from MULTI SCIENCES (Shanghai, China), following the manufacturer's instructions. Cells were collected after treatment with drugs for 48 or 72 h. Apoptotic cells were analyzed by flow cytometry after incubation with Annexin‐V FITC and propidium iodide (PI) for 30 min at room temperature using FACScan™ flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Cell cycle assay was also detected by flow cytometry using PI DNA staining from MULTI SCIENCES. After treatment with drugs for 48 h, the cells were harvested, fixed overnight with 75% ethanol at 4 °C, and then incubated with DNA staining for 30 min at room temperature. Analysis was conducted by FACScan™ flow cytometer (Becton Dickinson).

All strains were grown in YPD overnight at 30°C. Mitochondrial complex V activity was measured by an assay kit according to the manufacturer’s protocol. For determinations of intracellular ATP concentrations, an aliquot of 1 × 10⁶ cells from each strain was mixed with the same volume of BacTiter-GloTM reagent (Promega Corporation, Madison, WI, USA) and incubated for 5 min at room temperature as described previously (Zhang et al., 2013 (link)). ROS measurement was performed by using a oxidation-sensitive fluorescent dye DCFDA (Guo et al., 2014 (link)). Briefly, a suspension of 2.5 × 10⁶ cells was stained with DCFDA (20 μg/ml) at 37°C for 20 min. The emission spectra at 488 nm and excitation spectra at 595 nm were determined by FACScan flow cytometer (Becton Dickinson). The cyanine dye JC-1 was used for determination of mitochondrial membrane potential of each strain (She et al., 2015 (link)). A suspension of 2.0 × 10⁶ cells was incubated with 5 μM JC-1 at 37°C for 15 min and excitation spectra at 595 nm was determined by FACScan flow cytometer with emission spectra at 488 nm (Becton Dickinson).

A commercial Annexin V-FITC apoptosis detection kit (Sigma-Aldrich, St. Louis, MO, USA) was used to detect the ratio of apoptosis and necrosis. After the treatments, cells were collected and stained the Annexin V-FITC/propidium iodide (PI) reagents according to the manufacturer’s instructions. Stained cells were determined by FACScan flow cytometer (Becton Dickinson, CA) and analyzed with using ModFit software (Becton Dickinson, CA). The apoptotic and necrotic cells were presented as the percentage of total cell numbers. For determination of cell cycle arrest, the exponential growing A549 cells were treated with 3,5-DMAP at the indicated doses. The cells were permeabilized in 100% cold ethanol at -20°C for one day. After washing twice with cold 10% FBS, cells were centrifuged at 500 × g to remove debris. After washing with PBS, the cells were stained with propidium iodide (PI) solution (15 mg/ml PI and 2.5 mg/ml RNase A) (Sigma-Aldrich, St. Louis, MO, USA) and cell cycle phases were determined by FACScan flow cytometer (Becton Dickinson, CA). The cell cycle phase levels were presented as the percentage of total cell cycle phases (G1+S+G2/M).

To evaluate CD133 and CD44 surface expression 5 × 10⁵ cells were labeled with PE-conjugated anti-CD133 and FITC-conjugated anti-CD44 antibodies (see Supplementary Methods for antibodies details) for 15 min at 4 °C. Labeled cells were resuspended in Phosphate Buffer Saline (PBS)/0.5% Bovine Serum Albumine (BSA) and analyzed by FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) acquiring 10,000 events for each sample.
Analysis of apoptosis by flow cytometry nuclear DNA staining by propidium iodide (PI) was performed by a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) acquiring 20,000 events for each sample. The percentage of apoptotic cells was calculated in the sub-diploid region of the DNA content, registered as FL2 signals in linear scale.

Expression of CD14 and CD15 cell surface antigens, stained with PE-conjugated anti-mouse CD14 and CD15 antibodies (BD Pharmingen, San Diego, CA), was analyzed by flow cytometry using a FACScan Flow cytometer (Becton Dickinson, Bedford, MA) to control respectively monocytic and granulocytic differentiation, as previously described (22 (link), 29 (link), 30 (link)). FACScan Flow cytometer with Cell Quest software (BD) was used for acquisition and analysis. The results were expressed in terms of the percentage of positive cells and of the mean fluorescence intensity (MFI).