96 well transwell assay plates

Migration assays towards a 100 ng/ml CXCL12 gradient (#300-28 A, Peprotech) were performed using 96-well transwell assay plates (Corning Incorporated, Kennebunk, ME, USA) with 5-μm pore polycarbonate filters. First, 50 μl (0.75 × 105) SupT1 cells resuspended in assay buffer (RPMI supplemented with 0.1% BSA) were seeded into the upper chamber in the presence or absence of compounds and allowed to settle down for around 15 min. In the meantime, 200 μl assay buffer supplemented with or without 100 ng/ml CXCL12 as well as compounds were filled into a 96-well-V plate. Cells were allowed to migrate towards CXCL12 by putting the upper chamber onto the 96-well-V plates. After a migration time of 4 h at 37 °C (5% CO2), the lower compartments were analyzed for cell content by Cell-Titer-Glo^® assay (Promega, Madison, WI, USA). Percentages of migrated cells were calculated as described before^{45 (link)} and normalized to the CXCL12-only control.

Migration assays were performed using 96-well transwell assay plates (Corning Incorporated, Kennebunk, ME, USA) with 5 µm polycarbonate filters. First, 50 µl (0.75 × 10⁵) of SupT1 cells resuspended in assay buffer (RPMI supplemented with 0.1% BSA) were added into the upper chamber together with/without the compounds and allowed to settle down for around 15 min. In the meanwhile, 200 µl assay buffer with or without 100 ng/ml CXCL12 were filled into a 96 well-V plate. To avoid diffusion of the compounds from the upper to the lower compartment compounds were added in the same concentrations to the lower chamber. Next, the upper chamber was placed onto the 96 well-V plate and cells were allowed to migrate towards CXCL12 for 4 h at 37 °C (5% CO₂). Afterwards, migrated cells in the lower compartment were directly analyzed by Cell-Titer-Glo assay (Promega, Madison, WI, USA). The percentage of migrated cells was calculated as described by Balabanian et al. (2005)^{66 (link)}. To determine the relative migration in %, the percentage of migrated cells was normalized to the CXCL12 control without inhibitors.