Horse serum

GIFT was performed as previously described, with some modifications.17 (link),18 (link) Briefly, 6 mL blood samples from healthy individuals (C-1~6) were treated with ethylenediaminetetraacetic acid disodium salt (Wako Pure Chemical Industries, Japan), overlaid on 5 mL Lymphosepar I density gradient (Immuno-Biological Laboratories Company, Japan), and centrifuged at 2000 g for 10 minutes. After isolation of the granulocyte layer, erythrocytes were lysed in 10 mL of 0.19 M NH₄Cl lysis buffer at 37°C for 15 minutes. The cells were washed and resuspended in 1% paraformaldehyde (MERCK) in phosphate-buffered saline (PBS) and incubated for 10 minutes at room temperature. After three washes, 5 × 10⁵ cells were incubated with 50 µL sera [1:4 diluted in 2% horse serum (Cedarlane) in 0.1% NaN₃−PBS] in 96-well U-bottomed plates for 30 minutes at 37°C, followed by three washing steps and incubation with 50 µL fluorescein isothiocyanate-conjugated goat F(ab′)₂ anti-human IgG Fc Abs (1:100, Jackson ImmunoResearch Laboratories) for 30 minutes at 4°C. After two more washing steps, anti-granulocyte Abs were detected by flow cytometric analysis using an FC 500 MPL (Beckman-Coulter). The mode fluorescence intensity (MFI) of the cells gated for granulocytes in the scattergram was taken for data analysis.