Anti human cd300a pe clone e59

Cultured cells were stained for surface makers with monoclonal anti-human CD117-APC (clone 104D2, BD Biosciences, Erembodegem, Belgium), anti-human CD203c-PECy7 (clone NP4D6, eBioscience, San Diego, USA), anti-human FcεRI-PE (clone AER-37, Thermofisher Scientific, Waltham, USA), anti-human MRGPRX2-PE (clone K125H4, BioLegend, San Diego, USA), anti-human CD300a-PE (clone E59.126, Beckman Coulter, California, USA), anti-human CD32-PE (FLI8.26, BD Biosciences) and anti-human CD63-FITC (clone H5C6, BD Biosciences). Cells were stained for 20 min at 4°C in the dark. For staining of intracellular tryptase and chymase, cells were fixed with 1 mL of 4% paraformaldehyde solution for 30 min at room temperature, washed with and resuspended in PBS with 0.05% Triton-X-100 (pH=7.4) (Sigma-Aldrich). Next, cells were stained with monoclonal anti-human tryptase-Alexa Fluor 700 (antitryptase-AF700) (BioLegend) and monoclonal anti-human chymase-Alexa Fluor 700 (anti-chymase-AF700) (BioLegend), respectively, and incubated at 37°C for 30 min in the dark. Cells were washed and resolved in PBS with 0.1% sodium azide and analyzed. Representative plots of the immunphenotyping are shown in figure 1 of the online repository.

Cultured cells were stained for surface makers with monoclonal anti-human CD117-APC (clone 104D2, BD Biosciences, Erembodegem, Belgium), anti-human CD203c-PECy7 (clone NP4D6, eBioscience, San Diego, USA), anti-human FcεRI-PE (clone AER-37, Thermofischer Scientific), anti-human MRGPRX2-PE (clone K125H4, BioLegend, San Diego, USA), anti-human CD300a-PE (clone E59.126, Beckman Coulter, California, USA), anti-human CD32-PE (clone FLI8.26, BD Biosciences), anti-human CD63-FITC (clone H5C6, BD Biosciences) and anti-human CD25-FITC (clone B1.49.9, Beckman Coulter). Cells were stained for 20 minutes at 4°C in the dark, washed, and resuspended in PBS supplemented with 0.1% sodium azide (PBS_NaN3).
Additional staining for the basophilic markers CD123 and CD11b was performed to evaluate the potential presence of basophil-like cells 14, 34, 35 . In preliminary experiments, intracellular staining of trytase and chymase was performed, as previously described 27, 30 .