Streptomycin

MVEC were isolated from human adult foreskins using a previously described technique [17 (link),23 ]. In brief, foreskins obtained from adult circumcisions were placed in PBS supplemented with penicillin 200 units/mL, streptomycin 200 μg/mL and amphotericin B 0.5 μg/mL (all from Biological Industries, Kibbutz Beit Haemek, Israel). Foreskins were cut into 3 mm squares and placed in PBS containing 0.3% trypsin and 1% EDTA at 37°C for 30 minutes. Segments were then washed several times with PBS, placed in a Petri dish in M199 containing 10% foetal bovine serum (FBS), and individually compressed with the side of a scalpel blade to express microvascular fragments. The microvascular segments were passed through a 150 μm stainless steel mesh and collected by centrifugation at 300xg for 15 minutes. MVEC were seeded on a gelatin-coated cell culture flask and cultured in medium MCDB131 with 20% FBS; L-glutamine 2 mmol/L, penicillin 200 units/mL, streptomycin 200 μg/ml, EGF 20 ng/mL and bFGF 5 ng/mL (all from Biological Industries). When cells reached confluence, they were purified with Dynabeads CD31 (Dynabeads, Invitrogen Dynal ASA, Oslo, Norway) following the manufacturer’s instructions. Flow cytometry and positive staining for CD31, platelet endothelial cell adhesion molecule-1 (PECAM-1) confirmed the purity of the cell population. MVEC were used in passage 3

Mouse neuroblastoma N1E-115 cells (ATCC, Bethesda, MD, United States) were maintained in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 2 mM glutamine and 100 U/ml penicillin, 100 mg/ml streptomycin (Biological Industries, Beit Haemek, Israel). Human neuroblastoma SH-SYS5 cells (ECACC, Public Health England, Porton Down, Salisbury, United Kingdom; passage numbers from 14 to 16) were maintained in Ham’s F12: minimum essential media (MEM) Eagle (1:1), 2 mM Glutamine, 1% non-essential amino acids, 15% FBS and 100 U/ml penicillin, 100 mg/ml streptomycin (Biological Industries, Beit Haemek, Israel). Cells were incubated in 95% air/5% CO₂ in a humidified incubator at 37°C. Cells were differentiated with reduced FBS (2%) and DMSO (1.25%) containing medium (N1E-115 cells) or with retinoic acid at a concentration of 10 μM (SH-SY5Y cells) during 7 days before each experiment. Differentiated N1E-115 cells were treated for 2 or 4 h with SKIP/Ac-SKIP in final concentrations of 10^–12 – 10^–6 M, in the absence or presence of zinc (400 μM of ZnCl₂, stock solution – 0.1 M ZnCl₂ in water, Sigma, Rehovot, Israel).

RBL-MRGPRX2 cells were generated as previously described [24 (link)]. RBL-MRGPRX2 cells and their parental RBL-2H3 (herein referred to as RBL) counterparts were maintained at 37 °C, in a humidified incubator with 5% CO₂, in adherent cell cultures in low-glucose DMEM (cat # 01-050-1A, Biological Industries, Beit-Haemek, Israel) supplemented with 10% FBS (cat # 12657, GIBCO, Grand Island, NY, USA), 2 mM L-Glutamine (cat # 03-020-1A, Biological Industries), 100 μg/mL streptomycin and 100 U/mL penicillin and 12.5 U/mL nystatin (Biological Industries, Beit-Haemek, Israel), except for RBL-MRGPRX2 cells that were additionally supplemented with 1 mg/mL of G418 (cat # A1720, Sigma Aldrich, St Louis, MO, USA). LAD-2 cells (a kind gift from Dr. A.S. Kirshenbaum and Dr. D. Metcalfe, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA) were cultured in StemPro-34 (cat # 10640-019, GIBCO) supplemented with 1× StemPro-34 Nutrient, 2 mM L-Glutamine (cat # 03-020-1A, Biological Industries), 100 U/mL penicillin and 100 μg/mL streptomycin (cat # 03-032-1B, Biological Industries), and 100 ng/mL hSCF (cat # 300-07, Peprotech, Rocky Hill, NJ, USA).

RBL cells stably expressing N-terminally tagged hemagglutinin (HA) human MRGPRX2 (RBL-MRGPRX2) were previously described [12 (link)]. Cells were maintained as adherent cell cultures in low glucose DMEM (cat no. 01-050-1A, Biological Industries, Beit-Haemek, Israel) supplemented with 10% FBS (cat no. 12657, GIBCO, Grand Island, NY, USA), 2 mM L-Glutamine (cat no. 03-020-1A, Biological Industries), 100 μg/mL streptomycin, and 100 U/mL penicillin, 12.5 U/mL nystatin (cat no. 03-032-1B, Biological Industries), and 1 mg/mL of G418 (cat no. A1720, Sigma Aldrich, St. Louis, MO, USA), at 37 °C in a humidified incubator with 5% CO₂. LAD-2 cells (a kind gift from Dr. A.S. Kirshenbaum and Dr. D. Metcalfe, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA) were cultured in StemPro-34 (cat no. 10640-019, GIBCO) supplemented with 1× StemPro-34 Nutrient, 2 mM L-Glutamine (cat no. 03-020-1A, Biological Industries), 100 U/mL penicillin and 100 μg/mL streptomycin (cat no. 03-032-1B, Biological Industries), and 100 ng/mL hSCF (cat no. 300-07, Peprotech, Rocky Hill, NJ, USA).

All cell lines were stored in a humid incubator at a temperature of 37 °C and 5% CO₂. CT26 cells were grown in RPMI-1640 containing l-glutamine, supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml), nystatin (12.5 U/ml), sodium pyruvate (1 mM), and HEPES buffer (1 M) (Biological Industries, Kibbutz Beit Haemek, Israel).
DA3 cells were grown in Dulbecco’s modified Eagle’s medium containing 4.5 g/l d-glucose and 4 mM l-glutamine, supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and nystatin (12.5 U/ml) (Biological Industries).

Dental pulp tissues were obtained from the molars of five healthy patients undergoing orthodontic treatment. The extracted molars were kept in phosphate-buffered saline solution (PBS, Biological Industries, Kibbutz Beit Haemek, Israel) containing 100 U/mL penicillin, and 100 μg/mL streptomycin (Biological Industries, Kibbutz Beit Haemek, Israel). After they were transferred to the laboratory, the extracted molars were cut horizontally at 1 mm below the cementoenamel junction. The pulp tissues were gently separated from the crown and root and placed in a 100-mm Petri dish. The pulp tissues were cut into small pieces with a sterile blade and cultured in ɑ-MEM (Gibco, Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel), 100 U/mL penicillin, and 100 μg/mL streptomycin (Biological Industries). Tissue cultures were maintained in a humidified atmosphere of 5% CO₂ at 37°C. Cells from the fifth passage were used for the experiments.