Pe conjugated mouse antihuman icam 1

Following a 72-hour stimulation, the plate was placed on ice for 3 minutes. The cells were harvested by centrifugation and washed with ice-cold 1X PBS. Cell pellets were resuspended in PBS supplemented with 10% FBS and stained with 1 μg/mL phycoerythrin (PE)-conjugated mouse-antihuman ICAM-1 (BD Biosciences) or CD64 (Invitrogen) antibody and/or 1 μg/mL fluorescein isothiocyanate (FITC)-conjugated mouse-antihuman CD16 (Invitrogen) or CD89 (Invitrogen) antibody for 1 hour at 4ºC in the dark. Immediately after, the cells were washed 3 times with 1X PBS and analyzed using flow cytometry as described above. The desired population was gated based on light scattering properties and 10,000 gated events were collected. An unstained sample excluded auto-fluorescent cells from gating using SONY's automatic autofluorescence finder tool. A PE- or FITC-conjugated isotype control (Invitrogen) excluded non-specific antibody binding from the analysis. Cell death was measured by adding 1 μg/mL propidium iodide (PI) to each sample immediately before analysis. Mean fluorescence intensity (MFI) of the entire cell population was recorded.

Human umbilical vein endothelial cells were briefly washed with sterile HBSS (Gibco), detached from the plate using versene with 5% of trypsin-EDTA (0.025%, v/v), then washed with staining buffer (BD Pharmingen). Cells were then transferred to a FACS plate, washed and resuspended in 10% PE-conjugated mouse anti-human ICAM-1 and FITC-conjugated mouse anti-human VCAM-1 (both from BD Pharmingen) in staining buffer for 30 min on ice and in the dark. Then cells were washed, resuspended in HBSS and analyzed using a Guava EasyCyte flow cytometer (Merck-Millipore). Data analysis was performed using the Guava software (Merck-Millipore). Mouse IgG1κ-FITC and PE were used as isotype controls.