2.9. Chemotaxis Assays

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood taken from healthy volunteers. Ethical approval to obtain blood from healthy volunteers was granted by the Research Ethics Committee (12/NE/0121). PBMCs were isolated from heparinised blood using Lympholyte-H (Cedarlane Laboratories) as per manufacturer’s instructions.

Diffusion gradient chemotaxis assays were performed in a transwell system in 24-well companion plates (BD Falcon, Nottingham, UK) as described previously. Briefly, for PBMCs, 5 × 10⁵ cells were placed in the upper well of a 3 µm cell culture insert above (0–50 nM) WT or mut-CCL21 in 0.1% BSA/RPMI. The assay was incubated at 37 °C for 90 min. Migrated cells were quantified either by counting average number of cells per high power field or inserting them into FACS Tubes before CountBright™ Absolute Counting Beads were added. BD FACS Canto II Flow Cytometer was used to run the analysis. The number of migrated cells was calculated with the following formula:

For 4T1-Luc chemotaxis, fibronectin-coated 8 µm inserts were used. The undersides of filters were coated with 80 µL 2.5 µg/mL fibronectin (Sigma) for 30 min. Then, 2 × 10⁵ cells were placed in the upper well and allowed to migrate towards a range of chemokine concentrations for 6 h at 37 °C. Migrated cells were quantified by counting average number of cells/HPF.

In vitro, trans-endothelial chemotaxis assays were performed as above, except 72 h prior to the assay, 5 × 10⁴ HMEC-1 cells were cultured in 0.5 mL complete RPMI in cell culture inserts. All assays were performed in triplicate.