MCs were isolated from human foreskin tissue as previously described [51 (link)]. Each mast cell preparation/culture originated from several (2–10) donors to achieve sufficient cell numbers, as routinely performed in our lab [52 (link),53 (link),57 (link),88 (link),89 (link)]. The skin was obtained from circumcisions, with written, informed consent of the patients or legal guardians and approval by the university ethics committee (protocol code EA1/204/10, 9 March 2018). The experiments were conducted according to the Declaration of Helsinki Principles. Briefly, the skin was cut into strips and treated with dispase (26.5 mL per preparation, activity: 3.8 U/mL; Boehringer-Mannheim, Mannheim, Germany) at 4 °C overnight, the epidermis was removed, the dermis was finely chopped and then digested with 2.29 mg/mL collagenase (activity: 255 U/mg; Worthington, Lakewood, NJ, USA), 0.75 mg/mL hyaluronidase (activity: 1000 U/mg; Sigma, Deisenhofen, Germany) and DNase I at 10 µg/mL (Roche, Basel, Switzerland). Cells were filtered stepwise from the resulting suspension (100 and 40 µm strainers, Fisher Scientific, Berlin, Germany). MC purification was achieved by anti-human c-Kit microbeads (#130-091-332) and an Auto-MACS separation device (both from Miltenyi-Biotec, Bergisch Gladbach, Germany), giving rise to 98–100% pure preparations (FACS double staining of KIT/FcεRI (anti-FcεRI eBiosciene #11-5899-42), Fisher Scientific; anti-CD117 Miltenyi-Biotec # 130-111-593) and acidic toluidine blue (Sigma) staining, 0.1% in 0.5 N HCl (Fisher Scientific), as described previously [90 (link),91 (link)].
MCs were cultured in the presence of SCF, and IL-4 was freshly provided twice weekly when cultures were re-adjusted to 5 × 105/mL. MCs were automatically counted by CASY-TTC (Innovatis/Casy Technology, Reutlingen, Germany) [88 (link),92 (link)].
Experiments were performed 3–4 d after the last addition of growth factors. For inhibition studies, cells were pre-incubated with 666-15 (CREB inhibitor; 5 µM unless otherwise stated; from Merck Chemicals, Darmstadt, Germany) or SCH772984 (ERK1/2 inhibitor; 10 µM), Pictilisib (PI3K inhibitor; 10 µM), Trametinib (MEK1/2 inhibitor; 10 µM), SB203580 (p38 inhibitor; 10 µM), SP600125 (JNK inhibitor; 10 µM), Pimozide (STAT5 inhibitor; 10 µM) and STAT3-IN (STAT3 inhibitor; 10 µM), all from Enzo Life Sciences, Germany, or imatinib-mesylate (Gleevec, KIT inhibitor; 10 µM, from Biozol Diagnostica, Eching, Germany) or KT 5720 (PKA inhibitor; 2 µM, from Bio-Techne, Wiesbaden, Germany) for 15 min, then stimulated (or not) by SCF (100 ng/mL). IL-33 was purchased from PeproTech (Hamburg, Germany) and applied in a concentration of 20 ng/mL, as described previously [52 (link)].
MCs were cultured in the presence of SCF, and IL-4 was freshly provided twice weekly when cultures were re-adjusted to 5 × 105/mL. MCs were automatically counted by CASY-TTC (Innovatis/Casy Technology, Reutlingen, Germany) [88 (link),92 (link)].
Experiments were performed 3–4 d after the last addition of growth factors. For inhibition studies, cells were pre-incubated with 666-15 (CREB inhibitor; 5 µM unless otherwise stated; from Merck Chemicals, Darmstadt, Germany) or SCH772984 (ERK1/2 inhibitor; 10 µM), Pictilisib (PI3K inhibitor; 10 µM), Trametinib (MEK1/2 inhibitor; 10 µM), SB203580 (p38 inhibitor; 10 µM), SP600125 (JNK inhibitor; 10 µM), Pimozide (STAT5 inhibitor; 10 µM) and STAT3-IN (STAT3 inhibitor; 10 µM), all from Enzo Life Sciences, Germany, or imatinib-mesylate (Gleevec, KIT inhibitor; 10 µM, from Biozol Diagnostica, Eching, Germany) or KT 5720 (PKA inhibitor; 2 µM, from Bio-Techne, Wiesbaden, Germany) for 15 min, then stimulated (or not) by SCF (100 ng/mL). IL-33 was purchased from PeproTech (Hamburg, Germany) and applied in a concentration of 20 ng/mL, as described previously [52 (link)].
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