PKA inhibitor

FRC cell lines were established from peripheral LNs by long-term culturing as described previously^{74 (link)} with minor modifications. In brief, LNs from 8-week-old C57BL/6 N mice were dissected and disrupted using two 25 G needles before enzymatic digestion with DMEM medium containing 3.5 mg/ml Collagenase D and 40 μg/ml DNase I at 37 °C for 30 min with agitation⁷⁵ . The mixture was then filtered through a 70 μm cell strainer and centrifuged at 300 g for 5 min at 4 °C. The cell pellet was resuspended and cultured in DMEM medium (supplemented with 10% FBS and 1% Penicillin/Streptomycin) (5% CO₂, 37 °C). After 24 h, non-adherent cells were removed, and fresh medium was added to continue culturing until cells reached confluence. Adherent-stromal cells were then trypsinized, and triple-stained with antibodies to identify FRCs: Cd45 Pacific blue (1:100), Cd31 PE (1:100) and gp38 APC (1:100). FRCs were sort-purified using a MoFlo Optical Bench Sorter (Beckman Coulter) to achieve a purity of ≥95%. The sorted cells were immediately cultured in DMEM medium for expansion, and then seeded into 60 mm dishes in a density of 3 × 10⁵ per well to grow until confluence, followed by starvation for overnight and treatment with 10 μM isoproterenol (Sigma-Aldrich), 5 μM forskolin (Sigma-Aldrich), and 5 μM PKA inhibitor H89 dihydrochloride hydrate (Sigma-Aldrich) for 8 h. Culture media were collected to determine the concentration of IL-33 using a mouse IL-33 immunoassay kit (Immunodiagnostics Limited) or LDH using a CyQUANT LDH Cytotoxicity fluorescent assay kit (Thermo Fisher Scientific).

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)].

T0901317, 9-cis-retinoic acid, phorbol 12-myristate 13-acetate (PMA), GLP-1 (7–37), and exendin-(9–39) were purchased from Sigma (St Louis, MO, USA). Endotoxin, fatty-acid-free bovine serum albumin (BSA), and the PKA inhibitor 14–22 amide were purchased from Calbiochem (Merck KGaA, Darmstadt, Germany). Sitagliptin phosphate and vildagliptin were purchased from Toronto Research Chemicals Inc. (Toronto, ON, Canada).