Three laser lsr 2 flow cytometer

Cultures of Jurkat E6-1 (ATCC) and lentivirus transducted Jurkat E6-1 cells that express Zaire Ebolavirus glycoprotein on the surface (gift from C. Davis and R. Ahmed, Emory University School of Medicine) were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, HyClone) according to ATCC recommendations. Cells were washed with ice-cold FACS buffer (Dulbecco's PBS containing 2% FBS and 50 nM Dasatinib), counted, seeded at ∼50,000 viable cells per well in V-bottom 96-well plate for each mAb to be tested and incubated 60 min at 4 °C with serial tenfold dilutions of mAb in a total volume 100 μl per staining. Cells were washed with FACS buffer by centrifugation for 2 min at 800g followed by incubation with 1:500 dilution of secondary goat anti-human IgG PE Ab (SouthernBiotech) in FACS buffer. After washing, 5,000–10,000 live cell events were acquired using a three-laser LSR-II flow cytometer (BD Biosciences) and analysed with FlowJo software (Tree Star). The dead cell population was excluded using propidium iodide staining.

Apoptosis was assessed by measuring staining with annexin V. HUVECs transfected with miRNA inhibitors or mimics were collected with accutase (Biolegend, 423201), resuspended in 100 μl of annexin V binding buffer (Biolegend, cat 422201), after which PE-annexin V and of 7-AAD were added to each samples. After a 15 minute incubation at room temperature, 300μl of annexin V binding buffer were added per sample, and the samples were analyzed on a three laser LSR-II flow cytometer (BD Biosciences). Quantification of annexin V staining was performed in FlowJo v10 (Flowjo, LLC).