Epics elite

The apoptotic cells were quantified according to the instructions provided in an Annexin V-FITC (Sigma-Aldrich, St. Louis, MO, USA) cell apoptosis assay kit. Briefly, about 1.5 × 10⁵ cells were plated in 96-well plates and treated with galaxamide and its representative analogues (0, 2.5, 5, and 10 µg/mL) for 48 h. The cells were then resuspended in a 200-mL binding buffer. Afterward, 5 mL of annexin V-fluorescein isothiocyanate (FITC) was added, followed by incubation in darkness at room temperature for 10 min. The cells were again resuspended in 200 mL binding buffer and stained with 5 mL PI. The prepared cells were then analyzed using a flow cytometry (Coulter Epics Elite, Miami, FL, USA). The cells in the FITC-positive and PI-negative fractions were regarded as apoptotic cells.

The apoptotic cells were quantified using an Annexin V-FITC (fluorescein isothiocyanate) (Sigma-Aldrich, St. Louis, MO, USA) cell apoptosis assay kit according to the instruction provided with the kit [25 (link)]. In brief, about 1.5 × 10⁵ cells were plated in 6-well plates and treated with galaxamide and representative compound (0, 2.5, 5, and 10 μg/mL) for 48 h. The cells were resuspended in 200 mL binding buffer. Afterwards, 5 mL Annexin V-FITC was added and then incubated in darkness at room temperature for 10 min. The cells were again resuspended in 200 mL binding buffer and stained with 5 mL PI (propidium iodide). The prepared cells were then analyzed using a flow cytometry (Coulter Epics Elite, Miami, FL, USA). The cells in the FITC-positive and PI-negative fractions were regarded as apoptotic cells.

After incubation period, cells were collected by the trypsin treatment and fixed with 70% ethanol. The cellular DNA was stained for 30 min with 0.1 mg/ml propidium iodide solution. Finally, the cells were analyzed via flow cytometry (Epics Elite, Coulter, Hialeah, FL, USA).

Induced DCs from the upper chamber of each transwell were collected and suspended in 250 µL of PBS. DCs stained with the FITC-anti-CD1a antibody and R-PE-anti-SWC3a antibody (Beckman Coulter, Florida, USA) were re-suspended in PBS and analyzed using a flow cytometer (EPICS ELITE; Beckman Coulter, Florida, USA). Cells were stained with the FITC-labeled anti-SLA-DR antibody (AbD Serotec, Kidlington, UK) and the PE-labeled anti-CD80/86 antibody (Ancell, California, USA) for 30 min at room temperature in the dark. The cells were then rinsed with PBS, resuspended in PBS, and then detected by flow cytometry.
CD1a and SWC3a double-positive cells indicated the presence of DCs. Effects of PCV2-infected PIEC on CD80/86 and MHC-II expression on DCs were analyzed. In addition, the data collected from the flow cytometry experiments were used to determine the timing of the subsequent DC endocytosis and antigen presentation assays.

A human ovarian carcinoma cell line, ES-2, demonstrated the lowest expression level of MUC1 among all cell lines examined previously [24 (link)]; therefore, it was employed to investigate the effect of overexpression of MUC1. MUC1 cDNA encoding full-length MUC1 protein containing 22 or 42 tandem repeats, donated by Dr. O.J. Finn and Dr. M.A. Hollingsworth, respectively [36 (link),37 (link)], were ligated into pCEP4 vectors and stably introduced into ES-2 cells using standard electroporation procedures. After selection with hygromycin B (Calbiochem-Novabiochem Co., San Diego, CA, USA), cells exhibiting the highest levels (top 5%) of mAb MY.1E12 binding were obtained by sorting with an EPICS ELITE (Beckman Coulter Inc., Fullerton, CA, USA). After propagation in culture, cell sorting was conducted two more times. ES-2 cells transfected with cDNA encoding MUC1 protein containing 22 or 42 repeats were designated as ES-2/T-22 cells and ES-2/T-42 cells, respectively [15 (link)].