Neutrophils were freshly isolated from EDTA-blood from healthy donors by standard density gradient centrifugation using Ficoll (Lymphoflot, BioRad) followed by two cycles of hypotonic erythrocyte lysis with sterile water. Purified neutrophils were kept in PBS supplemented with 2 mM EDTA and 2% FCS at a concentration of 2 × 106 cells/ml and incubated with heat aggregated IgA at the indicated concentrations for 20 min at 4 °C. After washing, neutrophils were incubated with 1.25 µg/ml fluorescein isothiocyanate (FITC)-labeled goat F(ab)2 anti-human IgA (#2052-02; Southern Biotech) for 20 min at 4 °C.
Polystyrene beads (size 1 µm; Color Y; Estapor, Merck) were incubated with 100 µg/ml IgA in PBS for 1 h at room temperature and constant agitation. After washing, the beads were incubated with 1.25 µg/ml FITC-labeled goat F(ab)2 anti-human IgA (Southern Biotech) in PBS supplemented with 2 mM EDTA and 2% FCS for 20 min at 4 °C.
Flow cytometry was performed on a Gallios cytofluorometer and evaluated using Kaluza Analysis 2.1 software (both Beckman Coulter). Cell or bead aggregates, dead cells, and cell debris were excluded. Delta mean fluorescence intensity (ΔMFI) values were calculated by subtracting the MFI of neutrophils or beads stained with secondary antibody only.
Polystyrene beads (size 1 µm; Color Y; Estapor, Merck) were incubated with 100 µg/ml IgA in PBS for 1 h at room temperature and constant agitation. After washing, the beads were incubated with 1.25 µg/ml FITC-labeled goat F(ab)2 anti-human IgA (Southern Biotech) in PBS supplemented with 2 mM EDTA and 2% FCS for 20 min at 4 °C.
Flow cytometry was performed on a Gallios cytofluorometer and evaluated using Kaluza Analysis 2.1 software (both Beckman Coulter). Cell or bead aggregates, dead cells, and cell debris were excluded. Delta mean fluorescence intensity (ΔMFI) values were calculated by subtracting the MFI of neutrophils or beads stained with secondary antibody only.