Chemotaxicell

The chemotaxis assay was performed using a modified Boyden chamber assay as shown in Fig. 3A [9]. 10⁶ cells of dHL‐60 cells or mouse BM neutrophils were stimulated with or without 3 nm C5a for 4 h, and the culture supernatant containing the stimulated dHL‐60 cells or mouse BM neutrophils were added to the lower chamber. 10⁶ cells of THP‐1 or CD115‐positive mouse primary monocyte stained with BCECF (2 ´,7 ´‐bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein; Merck KGaA, Darmstadt) were added to a 3.0‐μm pore‐sized upper chamber (Chemotaxicell, Kurabo Industries Ltd.). After 30 min, the stained THP‐1 cells or CD115‐positive mouse primary monocytes were harvested from the lower chamber followed by cell lysis. In addition, the stained THP‐1 cells or CD115‐positive mouse primary monocytes were teated for the direct migration to C5a by adding C5a to the lower chamber. The fluorescence intensity was measured using a Perkin Elmer Plate Reader (ARVO^™ X3 Multilabel Reader) at excitation with emission wavelengths of 485 and 535 nm, respectively. The relative chemotaxis level of THP‐1 or CD115‐positive mouse primary monocyte was calculated using the fluorescence intensity under stimulation by a dHL‐60‐CS or Neut‐CS in the absence of C5a as 1.00.

Transwell chamber with filter membrane of 8 μm pore size (Chemotaxicell, KURABO Industries, Osaka, Japan) was coated with Cell matrix Type I-C (Nitta Gelatin, Osaka, Japan) for collagen coating. For invasion assay, collagen-coated filter membrane was treated with 120 μl of Matrigel (BD Biosciences, San Jose, CA, USA; diluted 1:10 in conventional culture medium) for 2 h at 37 °C before cells were seeded. LS174T control and each KD cells were seeded on each chamber (1.5 × 10⁵ cells in 200 μl medium) and 600 μl of medium was added with or without 50 μm Ebselen into the bottom of a 24-well plate. After 48 h of hypoxic culture condition (2% O₂) or normal culture condition (20% O₂), cells that had migrated through the filter pores were fixed in 10% formalin for 30 min. Next, cells were stained with Crystal Violet solution (0.1% Crystal Violet, 2% ethanol, PBS) and non-migrating cells were gently removed from the upper chamber with cotton. Ten random fields were scanned, and the intensity of stained cells was extracted using the optimal threshold parameters and calculated with LuminaVision image analysis software. Intensity of Crystal Violet staining was presented as the average of the calculated values per field with error bars. The experiment was repeated three times and a similar result was obtained.

The migration of HTR8 cells was assessed using a transwell system (Chemotaxicell; Kurabo, Osaka, Japan) equipped with 8 µm pore size polycarbonate filters. Cells were resuspended in their basal media containing 2% FBS and loaded into the upper compartment, which was coated in Matrigel (Corning, Inc., Corning, NY, USA). The transwells were then placed into 24-well culture plates containing basal media supplemented with 2% FBS for 48 h. The cells that invaded beyond the lower surface of the filters were fixed with cold methanol and stained with DAPI. In each experiment, the numbers of cells were counted in five randomly chosen microscopic fields per filter [32 (link)].