Fc 500 mcl flow cytometer

The method used was similar to that described previously [23 (link)]. Three groups of transfected BM-MSCs were seeded and cultured in a 96-well round-bottom plate at different density overnight. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by density gradient centrifugation and subsequently prestained with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE, C34554, Thermo Fisher Scientific). Accompanied by stimulation of 2 μg/mL phytohemagglutinin (PHA, L1668, Sigma-Aldrich), the stained PBMCs (5 × 10⁴ cells/well) were cocultured with three groups of transfected BM-MSCs at ratios of 1 : 2.5, 1 : 5, 1 : 10, and 1 : 20, with three replicates per groups. Five days later, PBMCs from different groups were collected and evaluated for cell division. For evaluating regulatory T cell (Treg) induction, T cells were separated from PBMCs by means of magnetic beads, cocultured with three groups of transfected BM-MSCs at a 1 : 10 BM-MSC : T cell ratio for 5 days (2 × 10⁵ T cells/well). Nonadherent cells were collected from the cocultures, and the proportion of Tregs present was evaluated by flow cytometry using the monoclonal antibodies anti-CD25-APC (302609), anti-Foxp3-PE (320107, BioLegend), and anti-CD4-FITC (555346, BD Biosciences). Staining was detected using an FC500 MCL Flow Cytometer (Beckman Coulter); 20,000 events were collected per sample.

All the analyses were performed in a FC500 MCL flow cytometer (Beckman-Coulter, CA, USA) with an air-cooled argon ion laser (488 nm, 15 mW). This standard instrument is equipped with two light scatter detectors that measure the forward scatter (an estimation of cell size) and the side scatter (an estimation of intracellular complexity), and five photomultiplier tubes that detect the appropriately filtered light. FITC fluorescence was collected at 525 ± 20 nm, PE at 575 ± 20 nm and 7-AAD at 675 ± 20 nm. When determining dead cells (for quantification of the cell surface expression of receptors) all measurements were restricted to live cells by gating the cells that excluded 7-AAD. In all other cases, the population was selected based on forward and side scatter parameters.

For flow cytometry analysis, RPE-1 cells were collected, washed once with cold phosphate-buffered saline, and then fixed in 70% ethanol. DNA was stained with 100 μg/mL propidium iodide and 50 μg/mL RNase A for 30 min at 37 °C. The samples were analyzed using an FC 500 MCL Flow Cytometer (Beckman Coulter).

For apoptosis assay, Annexin-V-FITC (fluorescein isothiocyanate)/propidium iodide (PI) staining was performed using Annexin-V-FITC/PI apoptosis detection kit (#65925; CST, USA) according to the manufacturer’s instruction. TPC-1 or K1 cells with different treatment were harvested at specified times and then resuspended in binding buffer. The cells were incubated with 5 µl of Annexin-V-APC and 5 µl of PI in the dark at room temperature for 15 min. Then, 400 µl of PBS was added to the mixture. The samples were analyzed by FC 500 MCL Flow Cytometer (Beckman, Brea, CA, USA)

To detect the surface markers of untransfected and three groups of transfected BM-MSCs, 2 × 10⁵ cells per tube were harvested and incubated with anti-CD73-APC (344005), anti-CD45-APC (368511), anti-CD11b-APC (301309), anti-CD19-PECY7 (302215), anti-CD34-PECY7 (343515), anti-CD90-PE (328109), and anti-CD105-PE (323205) antibodies (all from BioLegend) for 30 min at 4°C. Isotype control antibodies were used to assess nonspecific staining. Staining was detected using an FC500 MCL Flow Cytometer (Beckman Coulter); 20,000 events were collected per sample.