Arvo x3 multilabel reader

Intracellular iron levels were measured using a Metallo Assay LS Ferrozine Kit (AKJ Global Technology, Chiba, JAPAN) following the manufacturer’s instructions26 (link). Briefly, 5 × 10⁴ cells were lysed in 1 ml of RIPA buffer, sonicated using a Bioruptor UCD-200 (Cosmo Bio, Tokyo, Japan), and incubated in 0.1 M HCl for 30 min. After centrifuging the samples at 20,000 × g for 15 min, the iron levels were determined based on the ferrozine method54 (link) by measuring the absorbance at 570 nm using an ARVO X3 multilabel reader (Perkin-Elmer, Waltham, MA, USA).

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.