Anti ige

The results of basophil activation are expressed as the median fluorescence intensity (MFI) of the cells. The results of the cytometry were analyzed using the program FlowJo version 9.0 (TreeStar, Ashland, Ore). As a positive control for the assay, a tube with 200 μl of whole blood, 100 μl of phosphate-buffered saline (PBS), and 3 μl of anti-IgE (Biolegend) was used. As a negative control, a tube with 200 μl of whole blood and 100 μl of PBS was used.
The CD-63 and CD203c increase were expressed as the ratio of the median fluorescence intensity (MFI) obtained with the drug to the MFI obtained with the negative control. This ratio is called stimulation index.
The sensitivity of BAT was calculated as the number of positive tests in the patients group divided by all the patients. The specificity was calculated as the number of negative tests correctly classified in the control group divided by all controls times 100.

Isolated B cells were washed and stained with Live/Dead viability discrimination dye (Life Tech #L34968). Cells were subsequently blocked in 2% BSA supplemented with Fc Blocker (BioLegend) for 15 min and then stained with antibodies against B cell surface makers for 1 h [all antibodies were from BD Biosciences except CD38-PE/Texas Red (Life Technologies #MHCD3817); CD19-BV510, #562847; CD20-BUV396, #563782; CD27-BV421, #560448; IgD-APC, #348222; IgM-PerCP/Cy5.5, #314512; CD138-PE, #552026; IgG-PE/Cy7, #409316; CD45-APC/Cy7, #368516]. After staining, cells were washed two times in PBS without Mg⁺⁺ or Ca²⁺ and then fixed in 2% PFA before analysis on a BD Fortessa or BD LSR flow cytometer. Plasmablasts were defined as CD19⁺CD20⁺IgD⁻CD27⁺CD38⁺ B cells. B cell activation was determined with CD86 surface staining (#562432). Ig production was determined by intracellular staining with anti-IgA (Life Tech #Z25002), anti-IgE (Biolegend #325510), and anti-IgG antibodies in plasmablasts; AID was detected with antibody #Z25302 (Life Tech). For intracellular IRF5 staining, after overnight fixation, cells were permeabilized the following day in 0.1% Triton X-100 and rinsed in PBS 2× before blocking in 2% BSA solution. IRF5 staining was performed using anti-IRF5 antibody conjugated to Alexa Fluor 488 (Abcam Catalog#: AB193245).