Anti cd117 magnetic microbeads

Animal experiments were approved by the Animal Care and Use Committee of the Gwangju Institute of Science and Technology (GIST-2019-042). Mice were supplied by Damool Science (Daejeon, Republic of Korea) and Orient Bio, Inc. (Gyeonggi, Republic of Korea) To perform in vitro experiments, BMDM were isolated from the femur and tibia bones of C57BL/6 male mice. The bone marrow cells were collected from these bones using a 30 G syringe and ice-cold sterile PBS. The cells were then filtered through a 70-μm cell strainer to remove tissue debris and centrifuged at 300× g for 10 min at 4 °C. Red blood cells were eliminated from the pellet by adding 5 mL red blood cell lysis buffer for 5 min, and centrifuging the sample after adding 5 mL PBS. BMDM cells were maintained in 6-well ultra-low attachment plates with monocyte medium (RPMI1640 supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1× Glutamax (Gibco, Thermo Fisher Scientific), 1 mM sodium pyruvate, 1× non-essential amino acids, 10 mM HEPES, 55 μM β-mercaptoethanol, and 10 ng/mL M-CSF (Peprotech, Rocky Hill, NJ, USA)) at 37 °C for 5 d. Monocytes were purified by negative selection with anti-CD117 magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) using a magnet separator to remove non-monocytes from the cell population.

The MC line LAD2 was kindly provided from Dr. Arnold S. Kirshenbaum (National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA) and cultured as described (15 (link)).
Human skin mast cells (hsMCs) were isolated according to a published protocol (16 (link)) with modifications as reviewed and approved by the Charité—Universitätsmedizin Berlin Institutional Review Board. Human abdominal skin from healthy adults undergoing abdominoplasty was cut into strips and treated with Dispase Type I (Roche Diagnostics) at 2.4 U/ml and 4°C overnight. The dermis was cut into small pieces and digested with collagenase (Worthington) at 10 mg/g of tissue, hyaluronidase (Sigma-Aldrich) at 5–7.5 mg/g, and 10 µg/ml DNAse (Roche Diagnostics) suspended in 10 ml of dispersing medium per g (PBS, 1% penicillin/streptomycin, 5% FCS, 1.5 µg/ml Amphotericin B, 5 mM MgSO₄) for 1 h at 37°C. The isolated cells were separated from the remaining tissue by three steps of filtration (pore sizes 100, 40, and 20 µm) and the digestion was repeated one to two times. MCs were further purified from the suspension by positive selection using anti-CD117 magnetic microbeads (Miltenyi) and a magnetic cell sorter. MC purity in these separations typically exceeded 95%, as assessed by Kimura staining. Informed consent of the patients has been obtained.