Cd14 ecd

Cells were incubated with FcR blocking reagent for 5 min followed by incubating with or without the mixture of fluorescent-labeled antihuman monoclonal antibodies: CD14-ECD, CD15-PE-Cy5, CD3-fluorescein isothiocyanate (FITC), and IFN-γ-PE (Beckman Coulter, Brea, CA, United States) for 30 min. For intracellular staining, cells were treated with IntraPrep Permeabilization Reagent (Beckman Coulter, Brea, CA, United States) before the staining. The Annexin V-FITC and propidium iodide were used to eliminate the dead cells from the analysis. Auto-fluorescence and non-specific staining were determined by using isotype-matched controls. The analysis was performed using a Cytomics FC 500 flow cytometer (Beckman Coulter, Brea, CA, United States).

The PBMCs or neutrophils (10⁶ cells each) were washed and incubated with FcR blocking reagent (Milteny Biotec, Auburn, CA, USA) for 5 min, followed by incubation at room temperature for 30 min with or without the mixture of fluorescent-labeled anti-human monoclonal antibodies: CD14-ECD and CD15-PE-Cy5 (Beckman Coulter, Brea, CA, USA). The dead cells were eliminated from the analysis as deduced by staining with Annexin V-FITC and propidium iodide. The cell doublets were eliminated based on the size of the event (FSC). The analysis was performed using a Cytomics FC 500 flow cytometer (Beckman Coulter, Brea, CA, USA), and isotype-matched controls were used to determine auto-fluorescence and nonspecific staining.

In order to prevent unspecific binding of antibodies a 15 min blocking step with 100% FBS (heat inactivated, Gibco, Life Technologies, Zug, Switzerland) was performed before staining. Staining was done at optimally titrated concentrations with mouse anti-human antibodies specific for surface markers of interest - CD83-FITC, CD86-PE, CD80-PC7, CD14-ECD, CD11c-PC5 (all purchased from Beckman Coulter, Nyon, Switzerland) - and with corresponding isotype control antibodies for 20 min at 4°C in the dark. In order to remove unbound antibodies, cells were washed once with FACS buffer (PBS+1.0% FBS+0.05% sodium azide) before being finally re-suspended in FACS buffer containing fixative (Beckman Coulter, Nyon, Switzerland). Fixed cells were stored at 4°C in the dark until measurement.