Dispase 2 from bacillus polymyxa

Biopsies were obtained with the same model forceps in all patients. We followed previously published methods used in our laboratory for cell isolation with slight modifications for the esophageal epithelium.^{34 (link), 35 (link)} Briefly, four biopsies from each patient (two from the lower and two from the upper esophagus) were enzymatically digested in pre-warmed HBSS with CaCl₂ and MgCl₂ solution (Life Technologies, Grand Island, NY, USA) containing 150 U ml⁻¹ collagenase from Clostridium histolyticum (Sigma-Aldrich, St Louis, MO, USA), 100 μg ml⁻¹ dispase II from Bacillus polymyxa (Roche, Indianapolis, IN, USA) and 0.1 mg ml⁻¹ DNAse I (Sigma-Aldrich) for 30 min at 37 °C, spinning at 450 r.p.m. The digested tissue was filtered through a 70-μm nylon mesh cell strainer (BD Biosciences, San Diego, CA, USA). The remaining tissue in the strainer was mashed through the cell strainer and washed with culture media (RPMI 1640, Sigma-Aldrich) containing 1% L-glutamine, 10% fetal bovine serum, 1% non-essential amino acids, 1% sodium pyruvate, 1% antibiotics/antimycotic (Invitrogen, Carlsbad, CA, USA). Extracted cells were centrifuged at 1000 r.p.m., at 4 °C, for 5 min. The supernatant was discarded, and the pellet was resuspended in 1 ml of culture media. The isolated esophageal cells were counted using a Z1 Beckman Coulter Particle Counter (Beckman Coulter, Inc., Brea, CA, USA).

Mast cell isolation was performed as described previously.⁴¹ (link) Briefly, abdominal skin and subcutaneous fat tissues were obtained from patients undergoing abdominoplasty. Subcutaneous tissues and reticular dermis were removed surgically. Remaining tissues were minced and subjected to enzymatic digestion (2.4 U/mL dispase II from Bacillus polymyxa, Roche, Basel, Switzerland) at 4^°C overnight. After removal of the epidermis, dermal tissues were digested in collagenase I (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37^°C for 2 hours. CD117⁺ mast cells were enriched by magnetic cell sorting technology (MACS System, Miltenyi Biotec) as suggested by the manufacturer. To increase purity of isolated cells, the isolation procedure was repeated one more time using the CD117⁺ cell fraction of the first isolation run. Purity was tested by immunofluorescence and was >95 %. CD117⁺ mast cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 % (vol/vol) heat-inactivated foetal calf serum (both Gibco), 1 % (vol/vol) penicillin/streptomycin (Biochrom, Berlin, Germany), and 100 ng/mL recombinant human stem cell factor (PeproTech, Rocky Hill, NY, USA). Mast cells were stained with anti-tryptase antibody (#ab2378, RRID: AB_303023, Abcam, Cambridge, UK) according to a published protocol.⁴¹ (link)