Characterization of rabbit antiserum against FPRL1/LXA4R.
(A) Histogram of flow cytometry demonstrating binding of the rabbit antiserum (1:50; solid line) as compared with preimmune serum (same dilution; shaded curve). The original data of side scattering versus FITC (from the secondary antibody) are shown in dot plots below. Mean fluorescence intensities are 14 ± 3.31 with preimmune serum and 27 ± 2.41 with anti-FPRL1. Data presented are from 1 representative experiment of a total of 3. (B) RBL-2H3 cells, the stable transfectants RBL/FPR and RBL/FPRL1, and the HEK293-transient transfectants 293/FPRL1 were incubated with preimmune serum (1:50 dilution; shaded curve) or anti-FPRL1 (same dilution; solid line), followed by FITC-conjugated secondary antibody. The histograms of flow cytometry were shown. Data are representative of 1 of the 2 experiments. (C) Neutrophils were preincubated with either preimmune serum or anti-FPRL1 (1:50 dilution) for 30 minutes at 37°C, and loaded with Indo-1/am. Elevation of intracellular Ca2+ by 1 μM SAA stimulation was blocked by anti-FPRL1. The results are representative of 1 experiment of a total of 3. (D) Inhibition of IL-8 secretion. Neutrophils were preincubated with or without either preimmune serum or anti-FPRL1 (1:50 dilution each) for 30 minutes at 37°C, and then stimulated with 1 μM SAA for 4 hours. IL-8 secretion was measured by ELISA. The secreted IL-8 (1060 pg/106/mL) by 1 μM SAA without serum pretreatment was set as 100%. Data are presented as means ± SEM of 2 independent experiments each performed in duplicate.
