Methods
Materials and reagents Hypochlorous acid (HOCl) was used as a gel formulation (0.1% AFC, pH 5.5 ± 0.5) and was supplied as“PR022” by Realm Therapeutics, Inc. (Malvern, PA, USA). PR022 is a proprietary formulation of stabilized hypochlorous acid in a gelling agent and an emollient in development for the treat- ment of atopic dermatitis. In the Phase 2 clinical study, 2 concentrations of PR022 are being evaluated, 0.05 and 0.1% (NCT03351777). For active sensitization, house dust mite allergen (Dermatophagoides farina, GREER, Lenoir, NC, USA) was used. Poly-L-lysine, laminin, capsaicin, allyl iso- thiocyanate (AITC), 2-mercaptoethanol and mineral oil were obtained from Sigma (St. Louis, MO, USA). Dispase was purchased from STEMCELL Technologies Inc. (Cambridge, MA, USA). Fura-2-acetoxymethyl ester (Fura-2 AM), phosphate buffered saline (PBS), collagenase, chloroquine, histamine, and SLIGRL-NH2 were ordered from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Fetal bovine serum (FBS), Dulbecco’s modified eagle medium with L-glutamine (DMEM) and RPMI-1640 medium, Ca2+ and Mg2+-free hank’s balanced salt solution (HBSS), penicillin-streptomycin and fetal bovine serum (FBS) were from Mediatech Inc. (Manassas, VA, USA). MEM eagle (EMEM) medium was obtained from Lonza Group Ltd. (Allendale, NJ, USA). For determination of pro- tein content DCs protein assay kit was used (BIO-RAD, Richmond, CA, USA). The murine IgE ELISA (OptEIA) set was ordered from Becton, Dickinson and Company (Franklin Lakes, NJ). Recombinant mouse interleukin (IL-)1β, IL-31, and tumor necrosis factor (TNF)α were purchased from Pepro Tech, Inc. (Rocky Hill, NJ, USA). Enzyme linked immunosorbent assay (ELISA) kits for IL-1β, IL-4, IL-13, thymic stromal lymphopoietin (TSLP), thymus and activation regulated chemokine (TARC), and TNFα were obtained from R&D systems (Minneapolis, MN, USA). Serotonin and tofacitinib were ordered from Tocris Bioscience (Bristol, UK).
