Facscalibur system

Apoptosis was determined by cell cycle analysis. For analysis of the cell cycle profile, MCF-7 cells (5 × 10⁵ cells/60-mm dish) were cultured for 48 h in RPMI medium, and the cells were harvested after 12 or 24 h treatment with paroxetine by trypsinization and by centrifugation at 168× g (1000 rpm) for 5 min. The cells were then fixed in 70% (v/v) ethanol and washed with ice-cold PBS. Whole cells were incubated with propidium iodide (PI) staining solution (10 mM Tris (pH 8.0), 1 mM NaCl, 0.1% NP40, 0.5 μg/mL RNase, and 0.05 mg/mL PI) in the dark for 30 min at 37 °C. Cellular DNA content and apoptotic cells based on the PI signal and sub-G1 peak were measured using a FACSCalibur™ system (Becton Dickinson, San Jose, CA, USA) collecting 10,000 events per sample and analyzed using CellQuest Pro™ software (Becton Dickinson). For analysis of apoptotic and necrotic cell death, the cells were labeled using a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen™, San Diego, CA, USA). FITC-conjugated annexin V (5 µL) and PI (5 µL) were added in 100 µL of breast cancer cells (1 × 10⁵), and then incubated for 15 min at room temperature in the dark. Following incubation, the cells were analyzed using a FACSCalibur™ system (Becton Dickinson).

After starvation for 24 h, cells were treated with metformin for 24 h or transfected with the INHBA OE plasmid or siRNA for 48 h. We collected the cells and fixed them with ice-cold 70% ethanol for 4 °C overnight, and stored them at −20 °C until analysis. The cells were then dyed with PI/RNase Staining Buffer (BD Biosciences, NJ, USA) and assessed by a fluorescence-activated cell sorting (FACS) Calibur system (BD Biosciences). The Annexin V assay was performed using FITC Annexin V/Dead Cell Apoptosis kit (BD Biosciences, NJ, USA). CRC cells were seeded in cell culture dishes at a density of 5 × 10⁵ cells, incubated for 24 h, and subsequently treated with metformin or PBS for 24 h. The cells were collected and suspended in 1× annexin-binding buffer and stained with FITC and PI for 15 min at room temperature in the dark. the stained cells were analyzed by a FACS Calibur system (BD Biosciences).

CD54 levels on Jurkat cells were analyzed by direct immunofluorescence. Briefly, 1 x 10⁶ cells were washed with PBS, 0.1% fetal bovine serum (FBS) and then incubated for 30 min at 4°C with anti-CD54-PE (BD Biosciences) in a 1:50 dilution. Cells were washed again with PBS, 0.1% FBS and analyzed on a FACSCalibur System (BD Biosciences). Ten thousand events were measured per analysis and subsequently exported to FlowJo7 (Tree Star). QuantiBRITE reference beads (BD Biosciences) were resuspended in 500 μl PBS, 0.1% FBS and measured on a FACSCalibur System (BD Biosciences).

hADSCs were incubated with each antibody against human CD14, CD34, CD45, HLA-DR (FITC; BD), CD44, CD73, CD90, CD105, CD166 (PE; BD) and CCR2 (Alexa Fluor; Serotec) for 1 h at 4 °C. Corresponding mouse isotype antibodies were used as controls. The single color-stained cells were washed with PBS and fixed with 1% PFA in PBS. MSC immunotypes were determined by flow cytometry on a FACS Calibur System (BD) and the percentage of expressed cell surface antigens was calculated for 10,000 gated-cell events.
To quantify the percentage of iNSCs induced from hADSCs, iNSCs were incubated with NCAM-conjugated FITC, Nestin-conjugated PE (BD) and Ki67-conjugated FITC (eBioscience) for 1 h at 4 °C. Cells without antibody binding were used as controls. FACS experiment and data analysis were carried out according to manufacturer's instructions (FACS Calibur System, BD).

Apoptosis was evaluated by flow cytometry. Briefly, cells were stained with Annexin V-FITC conjugate and propidium iodide solution. A FACSCalibur system (BD Biosciences, United States) was used to analyze apoptosis.
The cells were fixed in 75% ethanol and stained with PI/RNase Staining Buffer (BD Biosciences). The cell cycle was analyzed by flow cytometry using the FACSCalibur system (BD Biosciences, United States).