Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) analysis

SELDI-TOF (Protein Chip System, Series 4000, Ciphergen, Freemont, CA) was performed to detect peptides of interest. CM10 Protein Chip Array (Ciphergen) was used to screen the culture medium of REC upon IL-1β activations. The steps for preparing CM10 chip are as follows: activation of spot surface with 100 mM Sodium Acetate (pH 4.0) for 10 min. After removing the solution, five microliters of sample were applied to the chip containing another 50 μl of 100 mM Sodium Acetate (pH4.0)/0.1% TritonX-100 and incubated for 60 min at room temperature (RT). Spots were washed one time with 200 μl of 100 mM Sodium Acetate, pH 4.0/0.1% Triton X-100 for 5 min at RT and two times (200 μl) of 100 mM Sodium Acetate, pH 4.0 for 5 min at RT and two times rapid washing with milli-Q water. Chips were dried in air-suction box for 10 min. Two times of 0.7 μl of Sinapinic Acid (SPA) was applied to each spot and air-dried naturally. Chips were read by SELDI-TOF instrument with 1200 nJ laser intensity.