Nystatin

VeroE6 cells were obtained from the American Type Culture Collection (ATCC^® CRL-1586TM; Summit Pharmaceuticals International). Cells were used and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), MEM non-essential amino acids, 2 mM L-Glutamine, 100 Units/mL Penicillin, 0.1 mg/mL streptomycin and 12.5 Units/mL Nystatin (Biological Industries, Beit-Haemek, Israel). Cells were cultured at 37 °C, 5% CO₂ atmosphere.

Vero E6 cells (ATCC^® CRL-1586TM) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), MEM nonessential amino acids, 2 nM L-glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin and 12.5 units/mL nystatin (Biological Industries, Kibbutz Beit Haemek, Kibbutz Beit Haemek, Israel). The cells were cultured at 37 °C in a 5% CO₂ and 95% air atmosphere.

Vero E6 (ATCC® CRL-1586^TM) were obtained from the American Type Culture Collection (Summit Pharmaceuticals International, Japan). Cells were used and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), MEM non-essential amino acids, 2 nM L-Glutamine, 100 Units/ml Penicilin, 0.1 mg/ml streptomycin and 12.5 Units/ml Nystatin (Biological Industries, Israel). Cells were cultured at 37 °C, 5% CO₂ at 95% air atmosphere.
ExpiCHO-S (Thermoscientific, USA, Cat# A29127) were used for expression of recombinant proteins as described above.

The murine mammary carcinoma 4T1 cells (ATCC^® CRL-2539™) were obtained from ATCC (Rockville, MD, USA). The cells were grown in RPMI-1640 with 100 units/mL penicillin, 100 µg/mL streptomycin, and 12.5 units/mL nystatin (Biological Industries, Beit Haemek, Israel), and incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. Cell viability assessment by the Hoechst assay was performed as described [42 (link)].

Whole intestines were removed from E17 chick embryos into DMEM-F12 medium (Sigma- Aldrich Company, Ayrshire. UK). Individual Intestines were washed and segmented into duodenum (pancreatic loop), cecum (two ceca were disconnected from the intestines at the ileo-cecal junction) and colon. Each segment was then added to a pool of identical segments. The pancreas was removed from duodenal loops, and loop segments were split lengthwise and sliced using a sterile scalpel blade. Slices were forced thru a stainless steel mesh (55mm diameter 100 micron opening, Sigma-Aldrich, Rehovot IL). The resulting fragments were collected, washed and suspended in complete DMEM-F12 medium containing: penicillin 100 U/ml, streptomycin 0.1 mg/ml, nystatin 12.5 U/ml, sodium pyruvate 0.11 mg/ml (all from Biological Industries, Beit Haemek, Israel), Glutamax™ 2 mM (Thermo-Fisher Scientific, Carlsbad, CA, USA), and BD™ Mito-serum Extender (BD Biosciences, Bedford, MA, USA. The resulting suspension was seeded into six well plates (Nunc), previously thinly coated with BD Matrigel matrix (Erembodegem, Belgium). The same procedure was applied to segments derived from ceca and colon. Plates were incubated at 37.5°C, 7.5% CO₂. Under these conditions, epithelial monolayers develop within 48h.

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).