Cellquest pro software 5

Briefly, transfected cells were collected and resuspended with phosphate-buffered saline (PBS). Then, the density of cells was adjusted to 1x10⁶ cells/ml. Transfected cells were double stained by propidium iodide and Annexin V-fluorescein isothiocyanate (20 mg/ml; BD Pharmingen; BD Biosciences) at room temperature for 10 min in line with the manufacturer's instructions. Finally, cell apoptosis was detected via flow cytometry using a FACScan^® (BD Biosciences) equipped with CellQuest Pro software 5.1 (BD Biosciences).

Surface expression levels of receptors were analyzed on THP-1 monocytes and different Mφ types using a FACSCalibur flow cytometer (BD Biosciences) and CellQuest Pro software 5.1 (BD Biosciences). Surface receptor expression levels were reported either as percentage of receptor-positive cells or as raw geometric mean fluorescence intensity (MFI) of receptor-positive cells. In addition, forward scatter (FSC) and side scatter (SSC) properties of cells were analyzed. A total of 10,000 single cell events were measured for each sample.

A549/DDP cells (3.5×10⁵ cells/ml) transfected with pEGFP/ID3 or pEGFP/control, or untransfected cells were washed with 1X PBS buffer and resuspended in 200 µl cell staining buffer (Beyotime Institute of Biotechnology, Shanghai, China). After blocking with 10% fetal bovine serum (FBS; HyClone Laboratories, Inc.) at 4°C for 1 h to minimize non-specific background signals, 5 µl allophycocyanin (APC)-conjugated anti-human multi-drug resistant protein-1 (MDR-1) antibody (ready to use dilution; cat. no. 348607; BioLegend, San Diego, CA, USA) was added for 10–15 min at 37°C in the dark. The expression of MDR-1 protein on the cell membrane was detected using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). CellQuest Pro software 5.1 (BD Biosciences) was used for acquisition and analysis of data.

Transfected and untransfected A549/DDP cells (3.5×10⁵ cells/ml) were seeded into 12-well plates and incubated with 0, 1.0 or 2.0 µg/ml DDP for 24 h. The A549/DDP cells were then washed with 1X PBS and resuspended in 100 µl binding buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Next, Annexin V-FITC and PI (Sigma-Aldrich; Merck KGaA) were added for 30 min at 37°C in the dark. Following dilution with 400 µl binding buffer, staining was analyzed within 1 h by flow cytometry. The fluorescence intensity (green, FL1-H; red, FL2-H) was measured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). CellQuest Pro software 5.1 (BD Biosciences) was used for acquisition and analysis of data.

An Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to detect apoptotic activity according to the manufacturer's instructions. Chondrocytes were seeded into six-well plates at a density of 1x10⁶ cells/well in DMEM containing 10% FBS. When the cells reached 60% confluence, they were transfected. After 48 h, the cells were digested with 0.5% trypsin and resuspended in 300 µl binding buffer containing 5 µl Annexin V-FITC and 1 µl propidium iodide solution (100 µg/ml). After incubation for 20 min in the dark at room temperature, the stained cells were analysed by BD FACS Calibur with CellQuest Pro Software 5.1 (BD Biosciences).

The serum-free supernatant from the EC-109, MCF-7 and SK-BR-3 cell cultures was collected and centrifuged at 850 × g for 10 min at 4°C (ThermoHeraeus Multifuge X1R; Thermo Fisher Scientific, Inc.) for detection of uPA (three samples from each cell line). Triple wells were used in each cell line. An ELISA was performed to determine the level of uPA secreted from the cells using an uPA ELISA kit (catalog no., ab108917; Abcam, Cambridge, MA, USA) The assay was performed according to the manufacturer's instructions. The extracellular level of uPAR was determined using a FACscan flow cytometer (BD Biosciences, San Jose, CA, USA) (3 samples in each cell line). The procedure is briefly described, as follows: 1×10⁶ EC-109, MCF-7 and SK-BR-3 cells were incubated with an anti-uPAR antibody (10 µg per 1×10⁶ cells) (catalog no., sc-13522; Santa Cruz Biotechnologies, Dallas, USA) for 2 h at 4°C. The cells were washed three times with PBS containing 0.05% Tween-20 to remove the unbound antibody and then incubated with a fluorescein isothiocyanate-conjugated secondary antibody (dilution, 1:500; catalog no., ab6785; Abcam) for 30 min at room temperature. The normal mouse IgG (cat. no., sc-2025; Santa Cruz) were used as the controls. The flow cytometry results were analyzed using a FACscan instrument (CellQuest Pro Software 5.1; BD Biosciences).

Hypoxia-treated AC16 cells were analyzed using Annexin V-FITC early apoptosis detection kit (Cell Signaling Technology, Inc.) according to the manufacturer's instructions. The stained cells were analyzed on a BD Accuri™ C6 Flow Cytometer (BD Biosciences), and apoptotic cells (%) were the percentage of cells in Annexin V⁺/PI⁺ and Annexin V⁺/PI⁻ quadrants analyzed on CellQuest™ Pro software 5.1 (BD Biosciences).