Annexin 5 fitc

The trophoblasts were harvested for observation apoptosis. Cells were washed three times with cold PBS and resuspended in 200 μl binding buffer that contained 10μl of annexin V-FITC (R&D) and 5μl propidium iodide (PI) (Sigma, St Louis, MO, United States) for 15 minutes at room temperature while avoiding any light exposure. After incubation, the cells were analyzed with a flow cytometer of EPICS XL Coulter (Beckman-Coulter, United States). All assays were repeated three times.

Cells were seeded in 6-well plates at a density of 5 × 10⁴ cells per well and treated with drugs at different concentrations or DMSO as a negative control. We analysed apoptosis and the cell cycle status of the cells by using Annexin V-FITC and propidium iodide (PI) (R&D) staining according to the manufacturer’s protocol.

Apoptosis was quantitatively gauged by detecting phosphatidylserine exposure on cell membrane with Annexin V staining [20 (link)]. Cells were simultaneously stained with Annexin V-FITC (25 ng/ml; green fluorescence, R&D Systems, Minneapolis, MN) and dye exclusion (propidium iodide, 20 mg/ml, red fluorescence). Data were obtained by flow cytometry analysis with FACS-SCAN (Becton-Dickinson, Heidelberg, Germany) FACS Canto in cell populations from which debris was gated out and analyzed.

UC-MSCs were dissociated with 0.25% trypsin/0.04% EDTA, washed twice with PBS, and plated on T25 plastic tissue culture flasks (Nalge Nunc International, Rochester, NY, USA) at a concentration of 1.5‐2 × 10⁵ cells/cm² in neurosphere culture medium containing DMEM/F12 with 20 ng/ml of both epidermal growth factor (EGF; PeproTech, London, UK) and basic fibroblast growth factor (bFGF; R&D Systems, Inc., Minneapolis, MN) and N2 and B27 supplements (Invitrogen, Carlsbad, CA, USA). The same protocol was also used for the generation of neurosphere from extensively passaged MSCs, namely, passages 10, 20, and 30. At any given timepoint, neurospheres were enumerated under a 100x microscope for 6 random fields. For the apoptosis assay, UC-MSC-derived neurospheres were digested with 0.25% trypsin/0.04% EDTA for 5 minutes at 37°C and continuous pipetting using 200 μl pipetting tips to ensure that a single-cell suspension was achieved under a microscope. After spinning down, dissociated neurospheres (10⁵ cells) were resuspended in 500 μl binding buffer. Then, they were incubated for 5 min at room temperature in the presence of 0.5 μg/ml Annexin V-FITC (R&D Systems, Inc.) and PI in binding buffer as described by the manufacturer. The percentage of apoptosis was determined by FACS.

ARPE-19 cells and HUVECs at 48 h post transfection were collected, washed with PBS, suspended in staining buffer, and incubated with Annexin-V-FITC (R&D, New Jersey, USA) and PI (R&D) per the manufacturers’ protocol. Flow-cytometric analysis was subsequently performed to identify apoptotic cells with a gallios flow cytometry (Beckman Coulter, Brea, USA). A total of 10,000 living cells were collected for each sample. Three groups of untreated ARPE-19 cells and HUVECs were included for scatter gating: (1) Unstained cells for cell selection and adjustment of photomultiplier voltage; (2) Annexin-V-FITC stained only cells for adjustment of the FITC channel; (3) PI stained only cells for adjustment of the phycoerythrin channel. Data were then displayed as two-color dot plot with Annexin-V-FITC (X axis) vs. PI (Y axis). Annexin-V positive cells were recognized as apoptotic cells.

Cancer cells were cultured in growth media, washed, and seeded in very low numbers (~ 10% confluence, 50,000 cells/well in a 6-well plate in 2 ml media, 5260 cells/cm²) in either SM or CM to simulate low cell numbers in micro-metastases. For adhesion studies, cells were imaged for 8 h, in 20 min intervals, using the IncuCyte system (Essen Bioscience). The number of adhered cells was determined. For apoptosis studies, cells were incubated in CM or SM for 24 h, then washed and stained for AnnexinV-FITC (R&D systems) and 7AAD (R&D systems). Using flow cytometry, the percent of live/dead/early apoptotic cells was determined.