Macrophage Migration Assay Using Boyden Chamber

For the in vitro MΦ cell migration assay, we used 24-well plates with polycarbonate (PC) Boyden chamber (8 μm pore; Corning^®, New York, NY, USA) as previously described [35 (link)]. We used the CM of the glomeruli from Ctrl, DN rats as a chemoattractant, and neutralizing antibodies against CCL2 (500 ng/mL; NBP1-07035; Novus Biologicals, Littleton, CO, USA), CCL3 (500 ng/mL; MAB66252; R&D Systems, Inc., Minneapolis, MN, USA), and CCL21 (500 ng/mL; AF457; R&D Systems, Inc., Minneapolis, MN, USA). The bottom of the transwell inserts were coated overnight with 15 μg/mL of bovine fibronectin. To isolate rat peripheral blood mononuclear cells (PBMCs), a protocol by flotation using a low density iodixanol (OptiPrepTM, Merck KGaA, Darmstadt, Germany; see Application Sheet C05 for details) barrier was used [35 (link)]. A total of 1 × 10⁵ rat MΦs were seeded into the top of the transwell inserts and 650 μL of CM was added in the bottom of the well. HAM-F10 and FBS (5%) were used as a chemoattractant negative and positive controls, respectively. Twelve hours later, MΦs in the bottom of the well were fixed with 70% ethanol for 10 min and stained using DAPI (300 nM) for 10 min. Five different quadrants were captured using 400× magnification in an epifluorescence microscope (Zeiss) and cells were counted using Image J software Version 1.53t (NIH, Bethesda, MD, USA).