Anti cd49b apc

Untouched human basophils were stripped of pre-bound endogenous IgD as described in published studies (Chen et al., 2009 (link)) and incubated in PBS with or without 50 μg/ml monoclonal IgD from human myeloma (Athens Research and Technology), 2.5 μg/ml galectin-9 (R&D Systems), or 50 μg/ml IgD premixed with 2.5 μg/ml galectin-9 on ice for 15 minutes. Following washing, basophils were sequentially stained with F(ab)2 to IgD biotin (Southern Biotech) and streptavidin (Life Technologies) along with antiFcɛRI FITC (clone: AER-37; eBioscience) and anti-CD44 PE (clone: IM7; Biolegend). Following additional washing, stained cells were fixed with 1 % paraformaldehyde, stained with 100 ng/ml DAPI (Sigma-Aldrich), and imaged. A similar strategy was used to image basophils included in splenocytes from Balb/c mice. These cells were incubated with an Fc-blocking 2.4G2 mAb (Tonbo Bioscience) and subsequently stained with anti-FcɛRI FITC (clone: MAR-1), anti-IgD PE (clone: 11–26c.2a) (BD Biosciences), anti-CD49b APC (clone: DX5) (Biolegend), and Ghost Violet 510 viability dye (Tonbo Bioscience). Following washing, stained cells were fixed with 1 % paraformaldehyde and stained with 5 μg/ml 7-AAD (Tonbo Bioscience).