The chemotaxis of mouse monocytes toward rat islet supernatants was evaluated using a 5-µm pore Transwell membrane (Corning Inc., Corning, NY, USA), as described previously.22 (link) The rat islet culture supernatants used in this assay were the same as those used to determine cytokine concentrations. MACS-enriched monocytes (1.0 × 105) were seeded in the upper chamber in 100 µL of serum-free medium, and 600 µL of islet supernatant or RPMI was placed in the lower chamber. Chemotactic activity is expressed as the mean number of migrated monocytes in 10 high-power microscope fields. Monocyte migration toward RPMI served as the negative control.
To validate the effects of MCP-3 on chemotaxis, anti-MCP-3 monoclonal antibody (mAb; 2 µg/mL; Abcam, Cambridge, UK) was added to the islet supernatant to neutralize MCP-3. Isotype-matched mouse immunoglobulin G (2 µg/mL; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) served as the control. To inhibit chemotaxis induced by MMPs, the MMP inhibitor GM6001 (10 μmol/L; Santa Cruz Biotechnology, Inc.) was used, as described previously.23 (link) GM6001 inhibits MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MT-MMP1, and MMP-26.
To validate the effects of MCP-3 on chemotaxis, anti-MCP-3 monoclonal antibody (mAb; 2 µg/mL; Abcam, Cambridge, UK) was added to the islet supernatant to neutralize MCP-3. Isotype-matched mouse immunoglobulin G (2 µg/mL; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) served as the control. To inhibit chemotaxis induced by MMPs, the MMP inhibitor GM6001 (10 μmol/L; Santa Cruz Biotechnology, Inc.) was used, as described previously.23 (link) GM6001 inhibits MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MT-MMP1, and MMP-26.
