Boyden chemotaxis chamber

HMVECs (BioWhittaker, Walkersville, MD, USA) were maintained in growth factor complete endothelial basal media (EBM) supplemented with 5% FBS. Cells were between passages 7 and 10, and did not display discernable phenotypic changes when observed before each assay. Cells were maintained at 37°C and 5% CO₂. HMVEC migration in vitro was tested using a modified 48-well Boyden chemotaxis chamber (Neuroprobe, Cabin John, MD, USA). HMVECs (1 × 10⁶ cells/ml EBM + 0.1% FBS) were plated in the bottom wells of the chambers with a polyvinylpyrolidone-free polycarbonate filter (8-μm pore size; Nucleopore, Pleasant, CA, USA). The chambers were inverted and incubated in a humidified incubator with 5% CO₂/95% air at 37°C for two hours, allowing HMVECs to attach to the membrane. The chambers were inverted again and Id1 was added at different concentrations, with PBS, or basic fibroblast growth factor (bFGF, 60 nM) used as negative and positive controls, respectively. After incubation for two hours at 37°C, the membranes were removed, fixed in methanol for one minute, and stained with Diff-Quick (VWR International, West Chester, PA, USA). Cell migration was determined in quadruplicate and analyzed in three high-power 40X fields per well. The experiment was performed four times. Data are expressed as the number of cells migrating per well.

Tests were performed using a Boyden chemotaxis chamber (NeuroProbe, Cabin John, MD, USA) with a polycarbonate membrane having a pore size of 5 micrometers. The migration stimulants MIP-3β (CCL19) and RANTES (CCL5) (in concentrations of 500 ng/ml and 50 ng/ml, respectively) (R&D Systems) were placed into the lower wells of the plate. A suspension of cells at a concentration of 1×10⁶ cells/ml was placed in the wells, with each well containing cells that were either untreated or pretreated with saliva for 18 hours at different concentrations (1:30 and 1:100 v/v) in the presence or absence of LPS (100 ng/ml) were placed into the upper part. The chemokine RANTES is specific for receptors present especially on immature DCs, including CCR5. The chemokine MIP-3β is specific for the CCR7 receptor and is present mainly in mature DCs. After a 1.5 hour incubation at 37°C in a humidified 5% CO2 incubator, the plate was disassembled and the membrane was removed, fixed and stained with Diff-Quik (Baxter Diagnostics, Düdingen, Switzerland). The analysis was performed by an optical microscopy lens with 100× magnification. Five different fields were counted per triplicate, making a total of 15 fields per treatment. The results are given in average ± SD number of migrated cells.