Facs aria 2 ow cytometer

Transfected cells (1 × 10 6 cells/well) were plated in 6-well plates. After treatment, cells were collected by centrifugation at 1500 rpm for 5 min and then incubated with 5 μL of FITC-conjugated Annexin V and 5 μL of PI for 20 min in the dark at 4 °C. The stained cells were detected by the BD FACS Aria II ow cytometer (BD Biosciences, USA).

A phenotype analysis of the macrophages was performed with a BD FACS AriaII ow cytometer (BD, USA) as previously described [16] . Brie y, the cells were labeled with CD11b-FITC, F4/80-PE, and CD206-APC (eBioscience, San Diego, CA) following the manufacturer's protocol. To analyze the prevalence of M2 macrophages, the cells were incubated with PE-conjugated F4/80 antibodies or APC-conjugated CD206. Flow cytometry was performed using a FACS CaliburTM ow cytometer, and the data were analyzed using Paint-A-Gate software (Becton Dickinson).

Transfected cells (1 × 10 6 cells/well) were plated in 6-well plates. After treatment, cells were collected by centrifugation at 1500 rpm for 5 min and then incubated with 5 μL of FITC-conjugated Annexin V and 5 μL of PI for 20 min in the dark at 4 °C. The stained cells were detected by the BD FACS Aria II ow cytometer (BD Biosciences, USA).

Segregated lung mononuclear cells were stained, subsequently, AMs (F4/80 + CD11c + ) were sorted using a FACS Aria II ow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The purity of separated cells was more than ninety-ve percent.

Sorting was performed on a FACS AriaII ow cytometer (Becton Dickinson) equipped with a 70 µm nozzle. 2 µm and 3.4 µm polystyrene beads (Spherotech) were used for size calibration. Before sorting, propidium iodide (PI) was added to the cell suspension to a nal concentration of 0.2-0.5 µg/ml. Then, pro les of dissociated cells from GFP-labeled strains were compared to pro les of cells from N2 worms to exclude auto-uorescent cells. Sorted cells were collected in 3 ml L-15/FBS medium in a 15 ml conical tube chilled on ice. For RNA extraction, sorted cells and whole worm control samples were collected by centrifugation in a swing-bucket centrifuge at 4400 rpm, 4 o C for 10 min. The supernatant was removed and 0.3 ml Trizol solution (Invitrogen) were added and stored at -80 o C. For culture, sorted cells were seeded onto a poly-D-lysine coated glass-bottom dish (MatTec) with daily changes of L-15/FBS buffer. Cells were visualized by confocal microscopy (Carl Zeiss, LSM780) with a 60 × oil lens.

Human peripheral blood mononuclear cells (PBMCs) of healthy donors were obtained from the CFAR Gene and Cellular Therapy Core Laboratory at UCLA, without identi cation information under federal and state regulations. Human monocytes were isolated from healthy donor PBMCs via magnetic-activated cell sorting (MACS) using human CD14 microbeads (Miltenyi Biotec, 130-050-201)followedby uorescenceactivatedcellsorting(FACS;sorted as hCD45 + hCD11b + hCD14 + cells) using a FACSAria II ow cytometer (BD Biosciences). Human A375 melanoma cells (10 x 10 6 cells per animal) and puri ed human monocytes (5 x 10 6 cells per animal) were mixed and s.c. injected into NSG mice to form solid tumours. Some experimental animals received i.p. injection of MAOI (phenelzine, 30 mg/kg/day) to block MAO-A activity. At the end of an experiment, tumour-associated immune cells were isolated foranalysis using ow cytometry.