The chemotaxis assay was conducted in HTS Transwell-96 well plates (Corning Incorporated) with 5.0 μm pore size polycarbonate membranes following the manufacturer’s instructions.
Total number of 0.3 × 106 MEC1 cells were seeded in the upper well of the transwell plate. Chemokine gradient was created by addition of CCL19 chemokine (R&D Systems, CCL19/MIP-3beta, 361-MI) in concentration of 100 ng/mL for D4476 dose response testing and 200 ng/mL in case of VANGL2-Venus or VANGL2 siRNA testing to the lower well of the plate. Sterile PBS/0.1% BSA solution in corresponding amount was used in control conditions. Cells were incubated for 6 hours and then number of migrated cells was analyzed by Accuri C6 Flow Cytometer (BD Biosciences). The assessment of cell viability was performed by TMRE staining (Tetramethylrhodamine ethyl ester perchlorate, Sigma-Aldrich) as described previously [10 (link)]. The migration index was calculated as the number of cells (treated or untreated) migrating in response to the chemokine divided by the number of cells migrating toward the control medium only. Graphs show either migration index or number of migrated cells in 20 μL of sample taken from the lower well of the transwell plate.
Total number of 0.3 × 106 MEC1 cells were seeded in the upper well of the transwell plate. Chemokine gradient was created by addition of CCL19 chemokine (R&D Systems, CCL19/MIP-3beta, 361-MI) in concentration of 100 ng/mL for D4476 dose response testing and 200 ng/mL in case of VANGL2-Venus or VANGL2 siRNA testing to the lower well of the plate. Sterile PBS/0.1% BSA solution in corresponding amount was used in control conditions. Cells were incubated for 6 hours and then number of migrated cells was analyzed by Accuri C6 Flow Cytometer (BD Biosciences). The assessment of cell viability was performed by TMRE staining (Tetramethylrhodamine ethyl ester perchlorate, Sigma-Aldrich) as described previously [10 (link)]. The migration index was calculated as the number of cells (treated or untreated) migrating in response to the chemokine divided by the number of cells migrating toward the control medium only. Graphs show either migration index or number of migrated cells in 20 μL of sample taken from the lower well of the transwell plate.