Reverse transcription (RT)-PCR was performed to detect mRNA for RANTES, eotaxin, eotaxin-2, eotaxin-3, MCP-3, MCP-4, and -actin

Reverse transcription (RT)-PCR was performed to detect mRNA for RANTES, eotaxin, eotaxin-2, eotaxin-3, MCP-3, MCP-4, and -actin. whereas eosinophils bound to IL-4Cstimulated HUVECs changed shape and transmigrated, becoming phase-dark. In some experiments eosinophils or HUVECs were treated with NBD-557 saturating concentrations of the specified antibodies for 10 min before assembly of the flow chamber. NBD-557 In other experiments, HUVECs were pretreated with 20 M PD 98059 or an comparative amount of DMSO for 30 min or with 10 M histamine for 10 min before assemble of the flow chamber. Finally, eosinophils (5 106/ml) were pretreated with 250 ng/ml pertussis toxin for 1 h at 37C. A dose response curve was performed to ensure that the pertussis toxin was used at saturation. The cells were washed twice with HBSS, resuspended at 5 105 cells/ml, and used in transmigration NBD-557 experiments. Transmigration Under Static Conditions. Eosinophils (5 105/ml) were drawn into the flow chamber, the flow was stopped, and the cells were observed for up to 20 min. Percent transmigration was assessed at the specified occasions using video microscopy as described in the previous section. Unlike transmigration under shear conditions, cells that had partially transmigrated frequently retracted their processes (see online supplemental material). This resulted in an overestimate of transmigrated cells under static conditions. Reverse Transcription PCR. Endothelial cells were stimulated with buffer alone or buffer made up of 20 ng/ml IL-4 for 24 h as described above. Reverse transcription (RT)-PCR was performed to detect mRNA for RANTES, eotaxin, eotaxin-2, eotaxin-3, NBD-557 MCP-3, MCP-4, and -actin. RNA was isolated using TRIzol according to manufacturer’s instructions. Reverse transcription was performed using First strand synthesis kit from GIBCO BRL according to manufacturer’s instructions. PCR was performed with QIAGEN Grasp Mix using 2 l of the RT reaction as template cDNA and the appropriate primer pairs. Primers pairs were designed based on published sequences. After 35 cycles, amplified PCR products were identified by electrophoresis as described previously (27). ELISA for CCR3-active Chemokines. Endothelial cells were stimulated with buffer alone or buffer made up of 20 ng/ml IL-4 for 24 h as described above. The supernatants from these cells were collected and ELISAs were performed to detect the presence of eotaxin, RANTES, or MCP-3 according to manufacturer’s instructions. ELISA kits are not available for eotaxin-3; therefore we developed a sandwich ELISA for eotaxin-3. Immunosorp plates from NUNC were coated overnight with anti-eotaxin-3 antibody (0.5 g/ml) in carbonate coating buffer (0.1 M Na carbonate, pH 9.2). The plates were washed four occasions with HBSS and supernatants or eotaxin-3 standard diluted in M199/A were added to the plate. After 2 h at 37C, the supernatants were discarded and the plates were washed four occasions before the addition of biotinylated anti-eotaxin-3 antibody (0.25 g/ml in HBSS/A). After an additional 2 h, the plates were washed four occasions and streptavidin-conjugated HRP at a 1:1,000 dilution in HBSS/A was added to the wells. Plates were again incubated for 2 h and washed four times before the addition of TMB one-step ELISA substrate. After 15 min the reaction was stopped by the addition of 1 M H3PO4 and the plates were read at 450 Casp-8 nm. Western Blotting and Cell Surface ELISA for Eotaxin-3. Endothelial cells were stimulated with buffer alone or buffer made up of 20 ng/ml IL-4 for 24 h as described above. Total protein expression in control and IL-4Cstimulated HUVECs was determined by Western blotting using an eotaxin-3 antibody to probe the membranes. Recombinant eotaxin-3 and recombinant MCP-4 were used as positive and negative controls, respectively. Surface expression of eotaxin-3 NBD-557 on control and IL-4Cstimulated endothelial cells was decided using a altered ELISA as described (27). Briefly, stimulated cells were washed once with ice cold HBSS and 2 g/ml anti-eotaxin-3 or an isotype matched nonimmune antibody in HBSS/A was added and incubated for 60 min on ice. Cells were washed three times with HBSS and then incubated with a 1:1,000 dilution of HRP-conjugated donkey antiCgoat IgG secondary antibody for 60 min on ice. Antibody binding was decided using TMB one-step ELISA substrate and quantified by measuring absorbance at 450 nm. Statistics. All experiments were performed at least three times. The data were analyzed using either unpaired Student’s test or by using analysis of variance (ANOVA) followed by the Bonferroni test for intergroup comparisons. values 0.05 were considered significant. Online Supplemental Material. Time-lapse images of eosinophils interacting with IL-4Cstimulated HUVEC are shown in online supplemental Videos 1C3. All videos were captured for 4C5 min at 30 frames per second and compressed to 15C20 s using Adobe Premiere..