Anti cd49b apc

Whole blood samples from all control and experimental groups were collected via cheek puncture at D63 to conduct a basophil activation test (BAT) according to the method described by Torrero et al. [18 (link)]. Briefly, whole blood was incubated (1.5 h at 37°C) with RPMI-1640 medium (Lonza), αIgE (125 ng/ml, eBioscience, Breda, the Netherlands), or whey (20 μg/ml, DMV International) to activate basophils. After red blood cell lysis (Whole Blood Lysing Reagents, Beckman Coulter, Fullerton, CA, USA), cells were stained with anti-IgE-FITC, anti-CD49b-APC, anti-CD4-PE, and anti-B220-PE (eBioscience) to select the basophil population while excluding T and B cells. Median fluorescence intensity (MFI) of activation marker CD200R-PerCp-eFluor 710 was determined with flow cytometry using a FACS Canto II (BD Biosciences, Alphen a/d Rijn, the Netherlands).

Blood was taken from the mice on day 56, stimulated, and analyzed for basophil activation as previously described by Torrerro et al.19 In summary, whole blood was diluted 1:1 using RPMI in heparinized tubes. After incubation with anti‐mouse IgE at 0.125 µg/mL (R35‐72, BD Biosciences) or PE at 20 µg/mL for 90 min at 37°C in 5% CO₂, activation was stopped with PBS containing EDTA in 12 × 75 mm test tubes. The Whole Blood Lysing Reagents was used to lyse the red blood cells and fix the samples according to protocol (Beckman Coulter, Fullerton, CA). To block the Fc‐receptor, cells were incubated with anti‐CD16/CD32 (clone 2.4G2) for 20 min. Cells were stained for 30 min at 4°C with the following fluorescent‐labeled antibodies: anti‐IgE‐FITC (1:100, clone 23G3), anti‐CD49b‐APC (1:200, clone CX5), anti‐CD4‐PE (1:200, clone RM4‐5), anti‐CD200R‐Percpefluor 710 (1:200, clone OX110), and anti‐CD19‐PE (1:200, clone 6D5) from eBioscience. Acquisition of the samples was performed on the BD Accuri™ C6 flow cytometer, analysis with BD sampler software (BD Biosciences). Fluoresence minus one technique was used to set the gates.