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.