Cell Culture
For all in vitro experiments, primary normal human epithelial keratinocytes (NHEK) in passage 3-6 where used. NHEKs were generated at the Universitäts-Hautklinik Tübingen, approved by the medical ethical committee of the Eberhard Karls University Tübingen (ethical approval: 547/2011BO2). The cells were isolated from juvenile foreskin derived from patients, who have given written informed consent. The epidermis was isolated using 50 mg/ml dispase II (Roche, Switzerland). Cells were separated using a trypsin-EDTA solution (Merck, Germany). The cells were cultivated in a humidified, 5 % CO2 atmosphere at 37 °C in CnT-07 medium (CELLnTEC, Switzerland) containing 10 μg/ml gentamycin and 0.25 μg/ml amphotericin B. The cells were kept under low calcium conditions (0.06 mM Ca2+). 24 h prior to treatment, the confluent cells were differentiated by adding 1.8 mM Ca2+. For experiments the cells were incubated with vehicle or mediators 1:50 in DMSO with final concentrations: CRAC inhibitor BTP-2 (Merk, Germany), 10 µM; PI4K inhibitor GSK-F1 (SYNkinase, Australia), 10 nM; PLC inhibitor U-73122 (Santa Cruz, USA), 4 µM; or IP3R inhibitor Xestospongin C (Abcam, USA), 2 µM for 1 h before IgG treatment.