Anti cd117 clone 2b8

Single cell suspensions of TECs were obtained collagenase (Collagenase D type IV, Worthington) and dispase (Roche) enzymatic dissociation as previously described [78 (link)] and thymocytes were obtained by pressing the thymus through a 70μm strainer (Fisher). Cells were stained with fluorochrome-conjugated antibodies in FACS buffer (PBS pH 7.2, 0.005M EDTA, 2% FBS) for 20 minutes on ice and washed. Propidium iodide (Invitrogen) was added (0.5 μg/ml) to each sample prior to analysis to exclude dead cells. Anti-CD326 (clone G8.8), anti-I-A/I-E (clone M5/114.15.2), anti-CD25 (clone PC61) and anti-CD117 (clone 2 B8) were purchased from Biolegend. Anti-CD44 (clone IM7), anti-CD8α (clone 53–6.7), anti-CD45 (clone 30-F11) and anti-Bcl-2 (clone 3F11) were purchased from eBioscience. Biotinylated anti-Ly51, anti-pStat3 (clone 4/p-Stat3 pY705) and anti-total Stat3 (clone M59-50) were purchased from BD Biosciences. Anti-CD4 (clone RM4-5) and Streptavidin Qdot 655 was purchased from Invitrogen. To exclude erythrocytes, granulocytes, dendritic cells, macrophages and NK cells from the thymocyte analyses, the following antibodies were purchased from eBioscience: TER-119, CD11c (cloneN418), CD11b (clone M1/70), NK-1.1 (clone PK136) and Ly-6G (clone RB6-8C5). Flow cytometry was performed on a Becton Dickenson FACS Aria II and data were analyzed using FlowJo software (Tree Star).

The surface expressions of FcεRI, CD117 (c-kit), ST2, β-Integrin and CD49b were analyzed by flow cytometry. Briefly, 1 × 10⁶ mast cells were washed with PBS and incubated with an anti-CD16/CD32 mAb (clone 32, Biolegend, London, UK) for Fc receptor blocking. The following primary, fluorochrome-coupled antibodies were used for analysis: anti-CD45.2 (clone 104, Biolegend), anti-FcεRI (clone MAR-1, Biolegend), anti-CD117 (clone 2B8, Biolegend), anti-ST2 (clone DIH4, Biolegend), anti-integrin-ß7 (clone FIB27, Biolegend) and anti-CD49b (clone DX5, Biolegend) and Live/Dead fixable aqua dead cell stain (Invitrogen Thermo Fischer, Munich, Germany). Cells were determined fully matured if >90% were FcεRI⁺CD117⁺ and only then further studies were conducted.

Bone marrow cells from C57BL/6 mice were filtered through a 70-μm cell strainer in PBS and neutrophils were isolated by negative MACS selection (Miltenyi Biotec) as described previously (Hasenberg et al., 2011 (link)) using biotinylated anti-TER119 (clone TER119, 1 μg/ml), anti-CD5 (clone 53-7.3, 2.5 μg/ml), anti-CD45R (clone RA3-6B2, 1.25 μg/ml), anti-F4/80 (clone BM8, 5 μg/ml), anti-CD117 (clone 2B8, 1 μg/ml) and anti-CD49b (clone HMa2, 2.5 μg/ml) antibodies (all from Biolegend).