Multiscreen mic

Chemotaxis experiments were performed in RPMI-1640 medium supplemented with 1% FBS using either ChemoTx (Neuro Probe, Gaithersburg, MD) or Multiscreen-MIC (5 µm pore size; Millipore, Billerica, MA) 96-well plates. The CCR4⁺ CCRF-CEM target cells were loaded in the upper chamber of the transwell plate and chemotaxis was performed in the presence of CCR4 ligands CCL17 or CCL22 at a concentration 3.5 nM (28 ng/mL) in the lower compartment. For characterization of the antagonistic properties, the cells were pre-incubated with IgG1 antibodies in the upper compartment. The chemotaxis plates were incubated at 37°C, 100% humidity, and 5% CO₂ for 3 hrs. The number of cells migrating into each lower compartment was quantified by flow cytometry using FACSCanto II (BD Biosciences). The number of migrated cells in the presence of the ligand alone was set to 100% migration and the numbers of migrated cells in the presence of ligands and antibodies were expressed as the corresponding percentage. The results were analyzed by fitting the experimental curves (% migrated cells vs. antibody concentration) by non-linear regression using a model ‘log [inhibitor] vs. response’ of the software program PRISM (GraphPad) and the corresponding IC₅₀ values were calculated.

THP-1 human monocytic cells were maintained in RPMI1640 supplemented with 10% fetal bovine serum, 50 U/ml penicillin, 50μg/ml streptomycin, 50 μM β-mercaptoethanol, and 20 mM HEPES (Invitrogen). Migration of THP-1 cells in response to CCL4 P8A was measured in a 96-well MultiScreen-MIC filter plate (Millipore) with RPMI medium without phenol red and supplemented with 0.1% BSA. THP-1 cells (75μl 1.5 × 10⁶ cells/ml) were placed in the filter plate. CCL4 P8A proteins in the presence or absence of IDE treatment were placed in the lower chamber at the indicated concentrations. After a 4-hour incubation at 37°C, the upper 96-well filter plate was removed and the cells that had migrated to the lower receiver plate collected and quantified by a luminescence ATP detection assay system (PerkinElmer Life and Analytical Sciences, Waltham, MA) and counted on a Tecan Safire 2 microplate reader.