In vitro culture of macrophages with ION

In order to characterize M1-type or M2-type macrophages, we examined the expression of specific surface receptors, such as iNOS or CD206 [26]. RAW 264.7 M0 macrophages were seeded in each well of a 48-well plate at a density of 7 × 10⁴ cells, followed by an overnight culture in DMEM at 37 °C. Afterward, the RAW 264.7 M0 macrophages were incubated with or without ION (40 μL, 4.3 nmole mL⁻¹) at pH 7.4 or 6.7 for a 3-day period. For the assessment of M1-type (iNOS⁺CD206⁻) or M2-type (iNOS⁻CD206⁺) macrophages, the ION-treated macrophages were fixed with 4% paraformaldehyde (PFA). Immunostaining was conducted using an anti-iNOS antibody to identify M1-type macrophages, and a mouse MMR/CD206 antibody to identify M2-type macrophages. Subsequently, immunofluorescence observation of iNOS and CD206 was performed using a donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 555, and a donkey anti-goat IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488, respectively. Confocal microscopy was employed for the visualization of all macrophages.

Furthermore, it is worth noting that lipopolysaccharide (LPS) stimulates the expression of inducible nitric oxide synthase (iNOS) in M1 macrophages, resulting in an enhanced production of nitric oxide (NO) [26]. To investigate this phenomenon, a total of 9 × 10⁴ treated macrophages were co-cultured with 3 × 10⁴ BNL-1MEA cells in individual wells of a 24-well plate, followed by incubation in culture medium. Subsequently, the culture medium was supplemented with 100 ng mL⁻¹ LPS and incubated for 24 h. The effects were assessed using the CellTiter 96® AQueous One Solution cell proliferation assay (Promega, Madison, WI). The untreated cells’ reduction of MTS was set at 100% and used as a reference, with the reduction in test cells expressed as a percentage relative to the untreated cells.