Chemotaxis assay

For the chemotaxis assay, neutrophils were diluted to a concentration of 4 × 10⁶ cells/mL in DMEM and 3% FCS and then treated with different concentrations (10^-6 M to 10^-9 M) of both the compounds for 10 minutes. The assay was performed using 3 μm pore size ChemoTx® System 96 well plates (Neuro Probe, Gaithersburg, MD USA) by adding 29 μL of 10^-6 M LTB₄ as a chemotactic stimulus or PBS as control to the bottom wells, and 25 μL of neutrophil-vehicle/compound suspension on top of the filter membrane. Plates were incubated for 3 hours at 37 °C with 5% CO₂. After 3 hours the filter membrane was removed, and migrated cells were manually counted by taking 20 μL from the bottom wells and diluting in a 1:1 ratio with trypan blue. Results were expressed as an average of total number of cells in the bottom well from samples run in triplicate.