Easysep mouse biotin positive selection kit 2

For scRNA-seq in Fig. 1, BMBAs generated by culturing bone marrow cells in the presence of IL-3 (0.3 ng/mL: BioLegend) for 7 days were subjected to scRNA-seq analysis. BMBAs generated from 5 mice were pooled to generate scRNA-seq data. For scRNA-seq in Fig. 2, single-cell samples were prepared from the bone marrow and spleen of Mcpt8^GFP transgenic mice and depleted of lineage-positive cells by using biotinylated lineage antibodies (anti-CD4, anti-CD8a, anti-CD19, anti-B220, anti-Gr-1 and anti-TER-119, dilution 1:400) and EasySep Mouse Streptavidin RapidSpheres Isolation Kit (Stemcell Technologies, catalog #: ST-19860). GFP-positive cells were sort-purified from lineage-negative cells. Basophils isolated from 5 mice were pooled to generate scRNA-seq data. For scRNA-seq in Fig. 5, single-cell suspensions were prepared from the bone marrow and infected skin and subjected to positive selection of CD49b-positive cells by using biotinylated anti-CD49b antibody (BioLegend; clone: DX5, catalog #: 108904, dilution 1:400) and EasySep mouse Biotin Positive Selection Kit II (Stemcell Technologies, catalog #: ST-17665) followed by the isolation of CD200R3⁺cKit^- cells by cell sorting. Basophils isolated from 5 mice were pooled to generate scRNA-seq data.

The spleen of 8w healthy female C57BL/6 mice was selected to prepare single cells and erythrocyte lysis. T cells were sorted by EasySep Mouse Biotin Positive Selection Kit II (Stemcell, 17663, U.S.A). In brief, the CD3+ T cells were marked by CD3-Biotin antibody (BioLegend, U.S.A) and connected with beads, adsorbed to the wall of the test tube in the magnetic field and washed down after discarding the supernatant, maintained in lymphocyte culture medium (Hyclone, U.S.A) containing 10% FBS and other culture conditions as previously described.

Monocytes were enriched from bone marrow cells by using EasySep Mouse Monocyte Isolation Kit (StemCell Technologies, catalog#: 19861) according to manufacturer’s protocol. Bone marrow monocytes (1 × 10⁶ cells/injection/mouse) or control PBS were intravenously administered to the mice once a day during the development of IgE-CAI.
For fate-tracking experiments and ex vivo monocyte culture experiments, monocytes were first enriched from the bone marrow of CD45.1 congenic C57BL/6 mice or BALB/c mice by using EasySep Mouse Monocyte Isolation Kit (StemCell Technologies, catalog#: 19861). Ly6C^hi monocytes were isolated from monocytes by using biotinylated anti-Ly6C antibody (clone: AL-21, catalog#: 557359, dilution 1:400; BD Biosciences) and EasySep Mouse biotin positive selection Kit II (StemCell Technologies, catalog#: 17665). Ly6C^lo monocytes were isolated from monocytes by depleting Ly6C⁺ population using biotinylated anti-Ly6C antibody (clone: AL-21, BD Biosciences) and EasySep Mouse Streptavidin RapidSpheres Isolation Kit (StemCell Technologies, catalog#: 19860).