Cytomics fc500 flow cytometry analyzer

To analyze the splenic and intratumoral T cells, the following antibodies were used: PE anti-CD3 (561799), PE anti-CD4 (561829), APC-Cy7 anti-CD8a (561967), FITC anti-CD44 (553133), Percp-Cy5 anti CD62L (560513), PE anti-TNF-α (561063), APC anti-CD45 (561018), APC anti-CD31 (561814), PE anti-Notch1 (562754), PE anti-Notch2 (562755), FITC Annexin V (556419), and propidium iodide (556463) were from BD Bioscience. APC anti-TCRβ (17-5961), PE-Cy7 anti-granzyme B (25-8898), PE anti-perforin (12-9392), and FITC anti-Ki67 (11-5698) were ordered from eBioscience. All antibodies were 1:100 diluted before use unless specified. For cell surface staining, cells were incubated in antibody-containing PBS for 15 min on ice before test on a Beckman Coulter Cytomics FC500 Flow Cytometry Analyzer. Dead cells were excluded with 1 μg/ml propidium iodide. For intracellular cytokine and Ki67 staining, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 90% ice-cold methanol for 30 min on ice and were then stained at room temperature for 1 h with corresponding cytokine antibodies. The data were analyzed using a Cell Quest Pro software. Cell sorting was done by a BD FACSAria cell sorter.

Human ES A673 cells were seeded in 6-well plates (5 × 10⁵ cells/well) and transfected with either 15 nM nCAR/miR-34a-5p, control RNA, or vehicle for 48 h. For cell apoptosis analyses, cells were incubated with Annexin V-FITC conjugate (5 μl), Annexin V binding buffer (400 μl) in a dark room for 15 min. For cell cycle analyses, cells were fixed in 70% cold alcohol for 1 h and stained with 0.5 ml PI/RNase for 30 min. Samples were analyzed on a Cytomics FC500 Flow Cytometry Analyzer (Beckman Coulter). The rate of increase is calculated following the formula: rate of increase = (apoptotic rate_miRNA − apoptotic rate_controlRNA)/apoptotic rate_controlRNA.