Goat anti-CRAMP Ab (M-13) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-CRAMP Ab were used as capture and detection antibody, respectively. Titration was performed with CRAMP peptide and a standard curve was constructed. Briefly, M-13 was coated onto an ELISA plate (Nalgene Nunc, Rochester, NY, USA) at a concentration of 0.5 μg/mL in PBS

overnight at RT. After two washes with PBST, the plate was blocked with 100 μL of Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 hr at RT. The samples at appropriate dilutions in triplicate were added to the plates along with the standard. The plates were incubated for 1 hr at RT, washed twice with PBST and then incubated for 1.5 hr with rabbit anti-CRAMP Ab (0.2 μg/mL) at RT. After two washes, an appropriate Selleckchem BMS-777607 dilution of HRP-conjugated AZD1208 goat anti-rabbit IgG F(ab’)2 (MP Biomedicals, Solon, OH, USA) was added, followed by incubation for 1 hr at RT. After two washes, the reagent 3,3,5,5-tetramethyl benzidine (Nacalai Tesque) was added as substrate/coloring agent. The absorbance was measured at 450 nm. The detection limit of the ELISA was 0.2 ng/mL. The supernatants of BALF obtained as described above were condensed by acetone precipitation and SDS-PAGE applied using Ready gels (4% T stacking gel and 10–20% T resolving gel) from Bio-Rad (Hercules, CA, USA). For Western blotting, proteins were electrotransferred from the gel to PVDF membrane

(Bio-Rad). Nonspecific binding was blocked by incubation of the membrane in Blocking One (Nacalai Tesque). Primary rabbit anti-CRAMP Ab was

used at a dilution 1:4000. Secondary HRP-conjugated goat anti-rabbit IgG F(ab’)2 (MP Biomedicals) was used at a dilution of 1:5000. Bands were visualized using an ECL Advance Western blotting detection kit (GE healthcare, Buckinghamshire, UK). Cathelin-related antimicrobial peptide antigens in neutrophils were detected by indirect immunofluorescence. In brief, BALF pellets prepared as described above were fixed on glass slides with methanol for 10 min at RT and washed with PBS for 10 min. The samples were incubated with 1 μg/mL of rabbit anti-CRAMP Ab and normal serum as control for 1 hr at RT. After washing with PBS for 10 min, Liothyronine Sodium they were incubated with a secondary Alexa Flour 488 goat anti-rabbit IgG Ab (Molecular Probes, Eugene, OR, USA) and Hoechst 33342 (Dojindo, Kumamoto, Japan) at 1 μg/mL each for 1 hr at RT. The cells were viewed on an inverted fluorescence microscope (Eclipse Ti; Nikon, Tokyo, Japan), and the images captured by using a CCD camera (Nikon digital sight DS-Qi1Mc). A mercury lamp was used for fluorescence excitation of Alexa Flour 488 (495 nm) and Hoechst 33342 (352 nm). BALB/c mice (5 weeks old) were intraperitoneally injected with 1 mL of thioglycolate broth (Nissui, Tokyo, Japan). Five hours later, exudate cells were harvested; approximately 90% of them were determined to be neutrophils by Giemsa staining. The neutrophils (5 × 105 cells) were stimulated with M.