Facsdivatm software version 6

Samples were prepared with 1 × 10⁶ cells in 100 ul PBS with 1 % FBS and incubated for 40 minutes at 4 °C with the following antibodies; fluorescein isothiocyanate (FITC) conjugated Anti-TRA-1-60 antibody (mouse IgM, FCMAB115F, Millipore, Billerica, MA, USA), FITC conjugated Anti-Human Embryonic Stem Cell Antigen-1 (HESCA-1) antibody (mouse IgM, FCMAB111F, Millipore) and FITC conjugated isotype mouse IgM (clone:11E10, eBioscience, San Diego, CA, USA). After incubation, cells were washed and analyzed with the BD LSR II Analyzer and BD FACSDiVaTM Software Version 6.1 (BD Biosciences, San Jose, CA, USA), or FlowJo 10.0.8 data analysis software (FLOWJO LLC, Ashland, OR, USA).

DU-145 cells were seeded in 6-well plates (5 × 10⁴ cells/mL; 2.7 mL per well) 24 h before the addition of assigned compounds/albumin NPs. At the time of drug treatment, stock solutions of compounds were diluted to 10-fold the desired final test concentrations in EMEM medium. Aliquots of 300 μL of these diluted drug solutions were added to the appropriate microtiter wells containing 2.7 mL of growth medium, resulting in the required final drug concentrations. The final concentration of DMSO (given that the compound required DMSO for solvation) in test culture was <1%. All cells were incubated in the dark throughout the 24–72 h exposure period and did not receive prolonged exposure to light. Following 24–72 h of exposure at 37 °C, cell apoptosis/cell cycle/mitochondrial depolarization were determined with Annexin V-FITC kit (Sigma Aldrich)/ MitoProbe™ DiOC2(3) Assay Kit (Life Technologies, Rhenium, Jerusalem, Israel), respectively, according to the manufacturer’s instructions. Samples were taken for measurement using a BD LSR-II Analyzer supported with BD FACSDiVaTM Software Version 6.1 for data analysis. Corrole fluorescence in binding analysis was measured using appropriate filters for excitation (405 nm, DAPI) and emission (<590 nm, Ds-RED).

Cells (DU-145) were seeded in 6-well microtiter plates (5 × 104 cells/mL; 3 mL per well) 24 h before the addition of assigned compounds/albumin particles. At the time of drug treatment, stock solutions of compounds were diluted to 10-fold the desired final test concentrations with EMEM medium. Aliquots of 300 μL of these diluted solutions were added to the appropriate microtiter wells containing 2700 μL of medium, resulting in the required final drug concentrations (according to assigned concentration). The final concentration of DMSO (given that the compound required DMSO for solvation) in test culture was <1%. All cells were incubated in the dark throughout the 24–72 h incubation period and were not exposed to light for prolonged periods. Following 24–72 h of incubation at 37 °C, cells were dissociated with trypsin, re-suspended in PBS pH 7.2, and pelleted at 3000 rpm for 5 min. Samples were washed 3 times with PBS, after which they were taken for measurement using a BD LSR-II Analyzer supported with BD FACSDiVaTM Software Version 6.1 for data analysis. Corrole fluorescence was measured using appropriate filters for excitation at 405 nm and reading emission at 660/20 nm.