Hts transwell 96 well permeable supports with polycarbonate membrane

CCR5 expressing A3.01 cells (A3.01-R5, 1.5 × 10⁵), pre-treated or not with PTX (100 ng/ml) during 3 hr, in prewarmed RPMI-1640 supplemented with 20 mM Hepes and 1% serum, were added to the upper chambers of HTS-Transwell-96 Well Permeable Supports with polycarbonate membrane of 5 μm pore size (Corning). PSC-RANTES (33.7 nM) or SDF-1 (control, 10 nM) was added to the lower chambers. Chemotaxis proceeded for 4 hr at 37 °C in humidified air with 5% CO₂. The number of cells migrating across the polycarbonate membrane was assessed by flow cytometry with Attune NxT flow cytometer (Thermo Fisher). Specific migration was calculated by subtracting spontaneous migration from the number of cells that migrated toward the chemokine.

Chemotaxis of A3.01 or A3.01R5 cells was determined using a Transwell system as previously described [79 (link)]. Cells (1.5 × 10⁵) in prewarmed RPMI-1640 medium supplemented with 20 mM HEPES and 1% human serum AB were added to the top chambers of HTS-Transwell-96 Well Permeable Supports with polycarbonate membrane of 5 μm pore size (Corning, New York, USA). The same medium with or without (spontaneous migration) CXCL12, PSC-CCL5 or bis-imidazoline compounds was added to the lower chambers. Inhibition assays of chemokine-mediated chemotaxis by AMD3100, MVC, P2G or bis-imidazolines were performed by adding inhibitors to both the upper and lower chambers. Cells were allowed to migrate for 5 h at 37 °C in humidified air with 5% CO2. The number of cells migrating across the polycarbonate membrane was then assessed by flow cytometry with a FACS CantoII (BD Biosciences).