Anti ige fitc

Whole blood was collected in a heparin and an EDTA tube. The EDTA tube was used for external CBC analysis. The heparin tube was kept at 18–25°C. Basophil identification was done using unstimulated blood (PBS) as well as blood stimulated with either Anti-IgE-FITC (Thermo Fisher, Waltham, MA) or fMLP (Sigma, St. Louis, MO). The samples were incubated for 20 min at 37°C followed by 10 min at 4°C (4 (link), 14 (link), 21 (link)–24 (link)). Each sample was stained with the following antibodies Anti-IgE-FITC (Clone Ige21), anti-CD193-PE (Clone 5E8), anti-CD123-PerCPCy5.5 (Clone 6H6), anti-CD203c-PECY7 (Clone NP4D6), anti-CRTH2-APC (Clone BM16), anti-CD3-AF700 (Clone UCHT1), anti-CD45-EF506 (Clone HI30) anti-FcɛRI-SB600 (Clone AER-37) (all Thermo Fisher, Waltham, MA) and anti-HLADR-Pacific blue (L243) (Biolegend, San Diego, CA) for 30 min at 4°C. Each antibody was titrated to obtain the best separation (25 (link)). The red blood cells were lysed using BD FACS lysis solutions (BD Bioscience, San Jose, CA) and resuspended in 400 μl PBS before acquisition.

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.