Optical borosilicate poly l lysine coated sterile glass covers

TMRM assay was performed using the probe in nonquenching mode conditions, as previously described in (Abramov, Fraley, et al., 2007). Cells were plated on 25‐mm optical borosilicate poly‐L‐lysine‐coated sterile glass covers (Thermo Fisher, Waltham, Massachusetts, USA) at a 70% confluence. The day after, cells were washed twice with HBSS and charged with 60 nM TMRM on HBSS. Cells were then incubated for 20 min in the incubator at 37°C and washed again with HBSS. After that, the medium was replaced by HBSS containing 15 nM of TMRM, to maintain the equilibrium distribution of the fluorophore. As shown in Supporting Information, Figure S1 under our experimental conditions, addition of FCCP leads to an abrupt drop in fluorescence. This confirms that TMRM fluorescence directly reflects the values of mitochondrial membrane potential and no appreciable quenching of TMRM fluorescence occurred. Cells were then mounted on Sykes‐Moore chambers (BellCo, Vineland, New Jersey, USA) and treated with the different CAIs and/or peptides. An image was taken at minute 0 and at minute 45, after adding the CAIs and/or peptides with different aggregation states.

SH‐SY5Y and microvascular ECs cells were plated on 15‐mm optical borosilicate poly‐L‐lysine‐coated sterile glass covers (Thermo Fisher, Waltham, Massachusetts, USA) at a 70% confluence. After 24 hr, cells were treated with the peptides in the presence or absence of MTZ or ATZ for 16 hr. Cells were then washed with PBS, fixed with 4% paraformaldehyde (10 min, RT), washed again, and blocked with 20 mg/ml BSA in PBS containing 0.3% Triton X‐100 (PBST). Slides were further incubated with mouse monoclonal anti‐CytC antibody (BD Biosciences; 1:200 in PBST containing 5 mg/ml BSA; 2 hr, RT) followed by Alexa Fluor 488‐conjugated anti‐mouse IgG (Thermo Fisher, Waltham, Massachusetts, USA) 1:200 in PBST with 5 mg/ml BSA for 1 hr at RT, as previously described fluorescence signals were visualized in a Zeiss LSM 510 laser scanning confocal/Confocor2 microscope using a 40× DIC oil immersion objective and LSM 510 software; acquired images were processed and analyzed using ImageJ (National Institute of Health).