Pe anti cd14 mab clone 61d3

Flow cytometry was used to access apoptotic/necrotic cells and determine nitric oxide (NOS) and reactive oxygen species (ROS) expression. At 24 and 48 hours, cultured cells were labeled with 5 µL PE–anti-CD14 MAb (clone 61D3; BD Bioscience). Afterwards, 5 µL of annexin V (AnV) FITC in annexin binding buffer (BD Bioscience PharMingen) was added for 20 min. Samples were analyzed on a FACS Fortessa flow cytometer (BD Bioscience PharMingen, San Jose, CA). To access NOS and ROS intracellular species, cells were labeled with dihydrorhodamine 123, DAF-FM diacetate (Life Technologies), dihydroethidium (Life Technologies), and CM-H2DCFDA (Invitrogen—Thermo Fisher Scientific), diluted according to the manufacturer’s instructions. Viable cells (AnV-/IP-) after 48 hours were counted. A minimum of 50,000 gated events from each sample were collected in a FACS Fortessa flow cytometer (BD Bioscience PharMingen, San Jose, CA) and analyzed using the FlowJo 7.6.5 program.

Flow cytometry was used to access apoptotic/necrotic cells and determine NOS and ROS species expression. After 48 h, cultured cells were labeled with 5 μL PE–anti-CD14 MAb (clone 61D3; BD-Bioscience Pharmingen, San Jose, CA). Afterward, 5 μL of the annexin V (AnV) FITC in the annexin binding buffer (BD-Bioscience Pharmingen) was added for 20 min. Samples were analyzed on a FACS Fortessa flow cytometer (BD-Bioscience Pharmingen, San Jose, CA). To access NOS and ROS intracellular species cells were labelled with dihydrorhodamine 123, DAF-FM diacetate (Life Technologies), dihydroethidium (Life Technologies) and CM-H2DCFDA (Invitrogen—Thermo Fisher Scientific), diluted according to the manufacturer’s instructions. Viable cells (AnV-/IP-) after 48h were counted. A minimum of 50,000 gated events from each sample were collected in a FACS Fortessa flow cytometer (BD-Bioscience Pharmingen, San Jose, CA) and analyzed using the FlowJo 7.6.5 program.