Chelex 100 beads

Primers, strains, and plasmids used in the current study are listed in Table S1 in the supplemental material. All primers for PCR were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). S. aureus was propagated in Bacto tryptic soy broth (TSB; BD, Sparks, MD) or on Difco tryptic soy agar (TSA; BD). To make TSB-c medium (40 (link)), TSB was stirred in the presence of 1% (wt/vol) Chelex 100 beads (Sigma-Aldrich, St. Louis, MO) for 4 h, a treatment that has been shown to reduce the level of available manganese to the low nanomolar range (40 (link)). The medium was then filter sterilized and supplemented with MgCl₂ and FeSO₄ to final concentrations of 50 µM and 1 µM, respectively. All antibiotics were purchased from Sigma-Aldrich and were used at the following concentrations for S. aureus: 5 µg/ml (gentamicin), 10 µg/ml (erythromycin), and 7 µg/ml (chloramphenicol).

To evaluate the role of dissolved metal ions in cytotoxicity and sensitization, KeratinoSens^TM cells were treated with solubilized metal ions followed by chelation, as described in our previous study, with slight modification (Jeong et al., 2018 (link)). Briefly, the stock solution of copper and cobalt chlorides in DW were incubated with Chelex 100 beads (Sigma-Aldrich) twice at 1:10 ratio (v/v) and shaken for 8 h at room temperature (26 ± 2°C). The beads were then removed by centrifugation at 15,000 × g for 30 min. The collected supernatant was diluted to the working concentration (2 mM) with cell culture medium and applied to KeratinoSens^TM cells.

To evaluate the role of dissolved metal ions in the cytotoxicity, cells were treated with solubilized ions followed by chelation, as described in our previous study with slight modification (Cho et al., 2012a (link)). Briefly, the stock solution of metal chlorides at 5 mg/mL in DW were incubated with 50 mg/mL Chelex 100 beads (Sigma–Aldrich) and mixed for 4 h at room temperature with continuous agitation. The beads were then removed by centrifugation at 15000 × g for 15 min. The collected supernatant was diluted to the final working concentration (100 μg/mL) with cell culture medium and treated to A549 or differentiated THP-1 cells.