Ferroorange probe

The cells (1 × 10⁵) were seeded on 96-well plates and treated with Ang II+Bleo for 5 days. The cells were incubated with 1 μM FerroOrange probe (Dojindo, Japan) with 1 mg/mL BSA for 30 min at 37 °C. After washing by D-Hank’s solution for three times, 100 μL of 1X D-Hank’s solution was added and the fluorescence intensity the excitation of 488 nm monitored under a fluorescence microscope digital microscope (Leica Microsystems, Berlin, Germany). The FerroOrange fluorescent intensity is proportional to the labile iron concentration.

After the EMP exposure, the Fe²⁺ levels in rat hippocampal tissues were determined using an Iron Assay Kit (MAK025, Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s protocols. The average ODs of the samples were measured using a SpectraMax 190-detection microplate reader (Molecular Devices, San Jose, CA, USA) at a wavelength of 593 nm.
HT22 cells were seeded on 8 chambered cover glasses (C8-1-N, Cellvis, Mountain View, CA, USA) and cultured with or without 1 μM Fer-1 (Selleck Chemicals, Houston, TX, USA) for 18 h before EMP exposure. After the EMP exposure, the intracellular Fe²⁺ concentrations of the HT22 cells were measured using a FerroOrange probe (F374, Dojindo, Kumamoto, Japan) following the manufacturer’s protocol. Fluorescence images of HT22 cells were observed using a confocal live-cell time-lapse imaging system (UltraVIEW VOX, PerkinElmer, Waltham, MA, USA).

Cellular and mitochondrial ferrous iron concentrations were assessed using the FerroOrange probe (Dojindo, Japan) and Mito-FerroGreen probe (Dojindo), respectively. Briefly, 5 × 10⁵ cells/well were seeded in 6-well plates and treated with 1 μmol/L SHK with or without DFOM. After 24 h, 1 μmol/L FerroOrange and 2 μmol/L Mito-FerroGreen probes were added to the cell culture, and the plates were incubated at 37°C for 30 min. The stained cells were monitored using a BD Accuri C6 flow cytometer (BD Biosciences). The tissue ferrous iron and total iron concentrations were assessed using an iron assay kit (Dojindo) according to the manufacturer’s instructions.

Cellular iron content was measured using the Iron Assay Kit (Sigma, MAK025) according to the protocol as described previously (Seibler et al., 2018 (link)). The absorbance at 593 nm was recorded using a Synergy H1MD plate reader (BioTek, United States). Ferrous iron (Fe²⁺) was assessed using FerroOrange probe (DojinDo, Japan) according to the instructions by the manufacturer. Briefly, the cells were transferred to a glass bottom cell culture dish (NEST, China; 801002) and cultured overnight in a 37°C incubator equilibrated with 5% CO₂, the supernatant was discarded, and the cells were washed with hank’s balanced salt solution (HBSS) three times, FerroOrange (1 μM) was added to the cells as HBSS solution, and cells were incubated for 30 min. Cells were observed under a Zeiss LSM710 Microscope with a 63 × 1.4 DIC Plan-Apochromat oil-immersion objective.

The supernatants obtained from HDFs’ lysates were incubated with a 2 μM FerroOrange probe (F374, Dojindo Laboratories, Kumamoto, Japan) at 37 °C for 30 min. The fluorescence intensity was then measured using a microplate reader (Synergy HT, BioTek) at an excitation/emission wavelength of 543/580 nm.

To prevent interference from Dmp1-GFP fluorescence in IDG-SW3 cells after mineralization induction, we used unmineralized IDG-SW3 cells for fluorescence staining. A C11-BODIPY probe (D3861, Invitrogen, USA) was used to evaluate cellular lipid peroxidation. After the indicated treatments, osteocytes were incubated with C11-BODIPY working solution (2.5 μmol·L⁻¹) for 30 min prior to analysis. A FerroOrange probe (F374, Dojindo, Shanghai, China) was used to detect intracellular Fe²⁺. After the indicated treatments, osteocytes were washed with PBS solution and treated with FerroOrange working solution (1 μmol·L⁻¹) for 30 min. Finally, stained cells were observed using confocal scanning microscopy.