Mouse anti human ige clone g7 26

To assess the ability of patients' serum to activate normal basophils, basophils from healthy donors were incubated with sera from patients, as previously described. 12 Briefly, 50 mL of buffy coat blood resuspended in activation buffer (containing 2 ng/mL of IL-3) was stimulated with 50 mL of patients' sera. A mouse antihuman IgE (Clone G7-26; BD Bioscience) was used as positive control. After 30 minutes at 37 C, cells were washed and stained for 30 minutes at 4 C with CD63 PE, CD123 PerCp-Cy5.5, HLA-DR PE-Cy7, and CD203c APC (all from BD Bioscience). Red blood cell lysis was performed next for 10 minutes at room temperature, with BD Pharm Lyse (BD Bioscience). Finally, samples were washed twice and acquired in a FACS Canto II flow cytometer (BD Bioscience). Doublets and dead cells were eliminated based on forward and side scatter parameters. Basophil populations were selected as CD123þ HLA-DR-cells, and the gate limit for CD63 positivity was established using the positive control tube. The basophil activation test (BAT) was considered positive when more than 5% of the total basophils were CD63-positive. The value of the 95th percentile of CD63þ cells induced by control sera was established based on previously published data. 13