Anti mouse cd49b percp

To confirm depletion of basophils (Supplemental Fig. 1), spleens were collected from male baso (−) mice (n = 2) and baso (+) mice (Basoph8, n = 2) at 4 d p.i. for flow cytometry. Spleens were passed through a 70-μm nylon cell strainer into 1% BSA in PBS. After RBC lysis (10× RBC lysis buffer multispecies, eBioscience), cells were pelleted at 500 × g for 5 min, washed once with 1% BSA, and counted. A total of 10⁷ cells per spleen were incubated with anti-mouse CD49b (PerCP) (eBioscience) and anti-mouse FCεRlα (allophycocyanin) (eBioscience) at the manufacturers’ recommended concentrations for 1 h at room temperature, protected from light, and then stained with DAPI (Invitrogen). Cells were then fixed for 20 min in 1% formalin, washed, and counted using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA). Live granulocytes were selected based on forward/side scatter and DAPI staining, and basophils within this population were defined as expressing both CD49b and FCεR1α (15 (link), 21 (link)). Data were analyzed with CytExpert software (Beckman Coulter).

To confirm depletion of basophil IL-18R (Supplemental Fig. 1), we collected spleens from basoIL-18R (−) mice (n = 3) and basoIL-18R (+) mice (n = 3) at 4 d PI for flow cytometry. Spleens were prepared as described previously (9 (link)). In brief, 10⁷ cells per spleen were incubated with anti-mouse CD49b (PerCP) (eBioscience), anti-mouse FCεR1α (allophycocyanin) (eBioscience), and anti-mouse IL-18R (PE) (eBioscience) at the manufacturer’s recommended concentrations for 1 h at room temperature, protected from light, then stained with DAPI (Invitrogen, Waltham, MA). Cells were counted using a CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA). Dead cells were excluded based on DAPI staining, and basophils within this population were defined as expressing both CD49b and FCεR1α (19 (link), 20 (link)). Data were analyzed with CytExpert Software (Beckman Coulter).