Human il 13

The concentrations of IL-6, IL-13, TNF-α, and histamine in supernatants of cell cultures were determined by ELISA. Kits for mouse IL-6 and TNF-α (eBioscience), mouse histamine (Oxford Biomedical Research, Rochester Hills, MI), mouse IL-13, and human IL-13 (R & D systems) were obtained from the indicated suppliers.

Human keratinocytes were isolated from neonatal foreskin as previously described [35 (link)]. PHK were cultured in Keratinocyte-SFM (Thermo Fisher Scientific, Waltham, MA, USA) with 1% Pen/Strep, 0.2% Amphotericin B (Thermo Fisher Scientific, Waltham, MA, USA). To differentiate PHK, cells were grown in DMEM (11965092, Thermo Fisher Scientific, Waltham, MA, USA) with 1% Pen/Strep, 0.2% Amphotericin B. For cytokine experiments, the following reagents were added alone or in combination to the culture media from the time of differentiation and replaced with every 48-h media change: human IL-4 (5–50 ng/mL; R&D system, Minneapolis, MN, USA), human IL-13 (5–50 ng/mL; R&D system, Minneapolis, MN, USA), human IL-17A (1–100 ng/mL; R&D system), JAK inhibitor I (10 μM; Calbiochem, San Diego, CA, USA), and PD98056 (10 μM; Calbiochem, San Diego, CA, USA). For the epidermal organotypic model experiment, keratinocytes were grown as previously described [36 (link)]. For all cytokine treatment studies, keratinocytes were starved of growth factors for 24 h before stimulation with the indicated cytokines.

The MAC-CYP cell generation was slightly modified from a previous report [25 (link)]. Briefly, after lysis of the red blood cells, bone marrow nucleated cells were collected and used for purification of CD11b⁺Gr1⁺ monocytes using the MACS technique. Gr1⁺ cells were positively selected using APC-conjugated anti-Gr1 antibody and anti-APC Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The positively selected cells were cultured in RPMI medium 1640 (Invitrogen) containing 10% FBS, human GM-CSF (R&D Systems), human G-CSF (R&D Systems), and human IL-13 (R&D Systems), each at 100 ng/mL for seven days. After seven days’ culture, the cells were transduced with indicated lentiviral vectors at a multiplicity of infection (MOI) of 5. Twenty-four hours later, the virus was removed, and culture media were replenished. The cells were cultured for another 24 h and examined for transduction efficiency under a fluorescence microscope. When necessary, the above transduction procedure was repeated one more time before use.