Chemotaxis Assays, Determination of Neutrophils Migration

Chemotaxis assays were performed using transwell filters (3 μm pore size). Neutrophils were suspended in HBSS/Ca⁺² (2 × 10⁵/100 μl) and placed in the upper compartment of the transwell chamber (Katayama et al., 1994) (Figure 6A).

Conditioned medium of HaCaT cells stimulated with FFA1 agonists increased chemotaxis of neutrophils. (A) Neutrophils were isolated from the blood of healthy donors and analyzed by flow cytometry prior to use in a transwell assay using the conditioned medium of HaCaT cells as a chemoattractant. (B, C) Neutrophil chemotaxis was analyzed using conditioned media from HaCaT cells treated with different concentrations of LA (10–100 μM) for 24 h. The data are represented as the number of neutrophils that migrated to the conditioned medium. The data are the mean ± SEM of five blood donors. *P < 0.05 and **P < 0.01, compared with the control, using Dunnett’s multiple comparison test. (D) Conditioned media of HaCaT treated with 10 µM GW1100 for 15 min and then stimulated with 50 µM LA for 24 h was used to evaluate neutrophil chemotaxis. The data are the mean ± SEM of five blood donors. *P < 0.05 and ***P < 0.001, compared with the control or linoleic acid-treated cells using Tukey’s multiple comparison test. (E, F) Neutrophil chemotaxis was analyzed using conditioned media from HaCaT cells treated with different concentrations of GW9508 (10–100 μM) for 24 h. The data are represented as the number of neutrophils that migrated to the conditioned medium. The data are the mean ± SEM of five blood donors. **P < 0.01, and ****P < 0.0001, compared with the control, using Dunnett’s multiple comparison test.

KCM was added to the lower well of the transwell plate at a 1:1 ratio with HBSS/Ca⁺², to a final volume of 600 μl. The plates were incubated at 37°C for 1 h, the time required for neutrophils to migrate through the polyvinylpyrrolidone-free polycarbonate filters (3μm pore size) which separated the upper and lower compartments (Katayama et al., 1994). Neutrophils that migrated across the filters were counted under a light microscope.