Ca am

On days 1 and 7, a live/dead assay was performed using calcein AM (CaAM, Sigma-Aldrich, 56496) as a marker for live cells and ethidium homodimer 1 (EthD1, Sigma-Aldrich, 46043) for dead cell nuclei. In 8-well μ-slides (ibidi), two replicate gels per condition were washed with NaCl/HEPES/CaCl₂ buffer and incubated in a staining solution with a CaAM (1 : 500) and EthD1 (1 : 1000) for 15 minutes at 37 °C. Subsequently, the samples were washed twice, the second time incubating for 10 minutes at 37 °C, and immersed in phenol red-free alpha MEM for imaging. At a Leica SP8 CLSM, three 100 μm z-stacks were taken per replicate. An automated analysis was conducted in ImageJ, analyzing particles in MIPs with live particle sizes >50 μm² and dead particle sizes limited to a range of 10–200 μm². The percentage of viable cells was calculated as the fraction of live cells over the sum of live and dead cells.

Intracellular labile iron concentration was determined by flow cytometry using the fluorescent iron sensor calcein acetoxymethyl ester (CA-AM). Briefly, cells were incubated with 0.25 μM CA-AM (Aldrich, Missouri, United States) for 30 min at 37°C in the dark. Then, cells were washed twice with PBS (1X) to remove the excess of CA-AM, and thus treated with 200 μM L1 (3-Hydroxy-1,2-dimethyl-4(1H)-pyridone, Sigma-Aldrich, Missouri, United States) or left untreated. The analysis was performed by FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). The difference in cellular fluorescence after and before incubation with L1 reflected the labile iron pool: $\Delta MeanFluorescenceIntensity,\Delta MFI={\Delta MFI}^{after}-{\Delta MFI}^{before}$

Cellular labile iron pool levels were measured with the use of calcein acetoxymethyl ester (CA-AM, Sigma-Aldrich, St. Louis, MO, USA) based upon protocols previously reported [57 (link),58 (link),59 (link)]. Monolayers of BLEC were treated in 96-well plates and simultaneously incubated with CA-AM (1 μM). Following staining, cells were analyzed by live imaging using an IncuCyte imaging system (Essen BioScience). Images were captured every hour and the increase in the green intensity fluorescence signal was calculated and analyzed by the IncuCyte integrated analysis software to quantify relative labile iron pool concentration.