Epics xl

After Ca9-22 cells were treated as above, the cells were collected, washed in PBS and treated with propidium iodide (PI). DNA contents were analyzed using an EPICS XL (Beckman Coulter). For Annexin V/PI staining, the treated cells were washed once with PBS and treated with the Annexin-V-FLUOS Staining kit (Roche Applied Science, Indianapolis, IN, USA) as previously described [7 (link), 13 (link)]. After incubation, treated cells were analyzed using an EPICS XL (Beckman Coulter), and apoptotic cell death was detected using the florescent nuclear dye Hoechst 33258 (Dojindo Laboratories Co.) and fluorescence microscopy.

Cultured U2OS cells that had been subjected to 24 h treatment (section 2.3) were fixed overnight in 70% ethanol at 4 °C. After detachment and centrifugation, cells were stained with 0.1 μg/mL RNase A (Beyotime) for 30 min at 37 °C and subsequently with 50 μg/mL propidium iodide (PI, Beyotime) for 30 min at 4 °C. Next, the cells were assayed by flow cytometry (EPICS XL, Beckman Coulter, Brea, CA) at λ_ex = 488 nm and emission in the FL3 channel. Each histogram was constructed on the basis of at least 5,000 events. Data were analyzed for cell cycle phases by calculating the percentage of cells in each phase using multicycle DNA cell cycle analysis software (FACScan, BD Biosciences).
Apoptosis was determined with an annexin V-FITC apoptosis detection kit (Beyotime) according to the manufacturer’s instructions. Briefly, U2OS cells were seeded into 6-well plates for 24 h and treated with the indicated chemical reagents for 24 h. After incubation the cells were collected, washed with PBS, resuspended in annexin V binding buffer, and incubated with annexin V-FITC/PI in the dark for 15 min. The cells were assayed by flow cytometry (EPICS XL, Beckman Coulter) at λ_ex = 488 nm and emission in the FL1 (FITC) and FL3 (PI) channels. The extent of apoptosis (propidium iodidepositive and annexin V-positive cells) was analyzed with FACS software (Beckman).

Non-adherent lymphocytes or purified CD4⁺ T cells were collected at 0, 1, 2 and 3 days following stimulation, and the cells were stained with phycoerythrin (PE)-conjugated anti-CD83, fluorescein isothiocyanate (FITC)-conjugated anti-CD4 or FITC-conjugated anti-CD25 (All from BioLegend, San Diego, CA, USA) and incubated on ice for 30 min in the dark. The controls were prepared using FITC- or PE-conjugated isotype controls. The intracellular staining of TGF-β-treated CD4⁺ T cells with anti-Foxp3 mAb was performed using an anti-human Foxp3 staining set (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions. The cells were subsequently analyzed using a flow cytometer (Beckman Coulter Epics XL; Beckman Coulter, Miami, FL, USA) by using a two-color acquisition method and the data were analyzed by using FlowJo 7.6.1 software (Treestar, Inc., Ashland, OR, USA).

Cell viability was evaluated by the MTT assay. Briefly, after treatment with Aβ₂₅₋₃₅, MSCs were seeded in 96-well plates and exposed to 10 μl MTT (5.0 g/l, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and incubated for 4 h at 37°C. Then, the medium was discarded, and 100 μl DMSO was added into each well. The formazan crystalline product was dissolved, and the optical density was measured at 570 nm with a microplate reader. Cell viability was expressed as a percentage of the value against the non-treated control group.
Apoptosis of MSCs was quantified using the Annexin V-FITC Apoptosis Detection Kit (KeyGEN BioTECH), according to the manufacturer's protocol. After exposure to Aβ₂₅₋₃₅, MSCs were washed with PBS, resuspended in 100 μl annexin V-FITC labeling solution, and stained with 5 μl annexin V-FITC and 5 μl propidium iodide (PI) for 30 min in dark at room temperature. Subsequently, samples were analyzed using flow cytometer (Beckman Coulter EPICS~XL, Beckman Coulter, Inc., CA, USA).