Keratinocyte preparation and narrow-band ultraviolet B irradiation
Normal human keratinocytes were isolated from neonatal foreskin and were grown in medium 254 (Cascade Biologics Inc., Portland, OR, USA) with human keratinocyte growth supplement (Cascade Biologics Inc.) and 1% penicillin-streptomycin-am- photericin B (10,000 U/ml, 10,000 pg/ml, and 25 pg/ml, repectively; GIBCO BRL., Grand Island, NY, USA) in a humidified atmosphere containing 5% CO2 at 37oC. Isolated keratinocytes were cultivated at 37oC and 5% CO2 in Epilife (Cascade Biologics, Inc., USA). Subconfluent primary cultures were passaged in secondary cultures, the tertiary passaged cells were used, and media was renewed every other day. During irradiation, culture dish lids were removed, and culture medium was removed and replaced with PBS (phosphate Buffered Saline, pH 7.4, GibcoBRL, USA). All experiments were independently repeated twice.
Keratinocytes were irradiated with NBUVB using a fluorescent lamp which emitted a peak wavelength of 312 nm (TL-01; Philips). Culture media was switched with PBS 72 h prior to irradiation in order to keep the cells in a quiescent phase. Conditioned media was returned to the dishes after UVB irradiation. Keratinocytes were exposed to various doses of irradiation (800, 1000 and 1200 mJ/cm2). The control group was maintained under the same conditions without UV irradiation. Irradiation doses were measured using a UV meter (Waldmann Medi- zintechnik, Schwenningen, Germany) for NBUVB.
Measurement of TGF- в s by ELISA
Culture media was harvested and stored at 80oC until ELISA was performed. TGF- в 1 and TGF- в2 were quantified using a human TGF- в ELISA kit (R & D Systems, Minneapolis, MN, USA). The absor- bance at 450 nm was determined in a microplate reader (E max; Molecular Devices, Sunnyvale, CA, USA). kamagra soft tablets
At the end of the final experimental period, total cellular RNA was purified from cultured cells by the RNA-Bee solution (Tel-test, Inc, Friendswood, TX, USA). The cells were lysed with 1.0 ml RNAzol B solution and extracted by adding 0.1 volume chloroform. After centrifuging at 12,000 g (4oC) for 15 min, the supernatant was transferred to a new 1.5 ml tube, and an equivalent amount of isopro- pranol was added. The samples were precipitated for 15 min at 4oC. After centrifugation, total RNA was measured at 260 nm using a spectrophotometer.