The mononuclear cells were

harvested and washed with HBSS, and 8 × 106 cells/well were allowed to adhere onto six-well tissue culture plates for 2 hr at 37° in serum-free RPMI-1640. Non-adherent cells and contaminating platelets were carefully removed from the plate by multiple wash steps using HBSS. The purity of cells remaining on the plate after 2 hr of adhesion was > 90% monocytes, with contaminating cells being platelets and lymphocytes. The remaining adherent cells were cultured overnight in RPMI-1640 containing 5% FBS. For studies using monocytes, adherent cells were washed and incubated in serum-free RPMI-1640 in the presence or absence of cytokines Sirolimus datasheet for 24 hr. In control experiments, purified lymphocytes or platelets were stimulated with IL-4 for 24 hr and the expression of CCL26 was determined. Neither cell type showed an increase C59 wnt chemical structure in CCL26 (data not shown). For MDM cultures, fresh RPMI-1640 containing

5% FBS and 5% human serum was added to the monocyte cultures after the overnight incubation. The cells were cultured for an additional 7 days to allow their differentiation into macrophages. Human serum, which contains monocyte colony-stimulating factor, was used to differentiate monocytes into macrophages as opposed to exogenous cytokines, as previously described by our group.14 Differentiation was determined morphologically, by flow cytometry, showing expression of CD14, but not CD83 (a dendritic cell marker), and by immunohistochemistry Interleukin-2 receptor examining CD14 and CD83 (data not shown). Following stimulation, U937 cells were lysed with hot 2 × Laemelli buffer. Proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), and Western blotting was performed using phospho-specific STAT6, total STAT6 or β-actin antibodies. Immunoblots were visualized using a Fluor-S MAX™ MultiImager and analysed

using quantity one software (Bio-Rad Laboratories, Hercules, CA). Total RNA was extracted from cells, and first-strand complementary DNA (cDNA) was synthesized using Superscript II, as described in the manufacturer’s instructions. cDNA was amplified by PCR using either Taq polymerase or TaqMAN Universal master mix. Primer sequences for standard PCR amplification were as follows. CCL26 forward primer: 5′-AGTCACAATTGTTTCGGAGTT-3′ reverse primer: 5′-AGTCTCCACCTTGGAACTG-3′ β-actin forward primer: 5′-CATGGATGATGATATCGCCG-3′ reverse primer: 5′-ACAGCCTGGATAGCAACGTA-3 Primer sequences for real-time PCR were as follows. CCL26 forward primer: 5′-ACACGTGGGAGTGACATATCCA-3′ reverse primer: 5′-GACTTTCTTGCCTCTTTTGGTAGTG-3′ probe: TACAGCCACAAGCCCCTTCCCTGG. A commercially purchased primer and probe were used for 18S ribosomal RNA (rRNA). The amount of CCL26 mRNA in each sample was calculated using the −delta delta Ct (−ddCt) method. Following stimulation, supernatants were harvested and stored at −20°.