In cell investigator v1

Necrotic cells were analyzed using the HCS platform In-Cell Analyzer 2200 (GE Healthcare) with PI (Thermo Fischer Scientific) dye, following the manufacturer’s instructions. The experimental design and treatments were performed as described for the apoptosis assay reported above. Along with the treatments, PI (10 μg/mL in PBS) and Hoechst 33342 (10 μM in PBS) were added to each well, and the samples were analyzed at 24, 48, and 72 h of treatment. The acquisition parameters were “brightfield” (Channel 1), Cy3 (Channel 2; PI), and DAPI (Channel 3; Hoechst 33342). The “autofocus” was set to “brightfield,” and the plates were scanned with acquisition mode “one field of view per well” in the 4× objective. The acquired images were analyzed using the In-Cell Investigator v1.3 software (GE Healthcare). Necrotic cells were quantified using the “average fluorescence intensity” recorded on the Cy3 channel. Representative images from DU145 spheroids were acquired by merging Hoechst (Blue/DAPI) and PI (Orange/Cy3). All analyses were performed with four spheroids/replicates (n = 4) in three biological experiments (n = 3).

All images for mitochondrial redox status analysis were acquired on the HCS platform In-Cell Analyzer 2200 (GE Healthcare) using the MitoSOX™ Red Mitochondrial Superoxide Indicator reagent (Cat. Nº. M36008; Invitrogen), following the manufacturer’s instructions. The experimental design and treatments were performed as described for the apoptosis assay reported above. Along with the treatments, MitoSOX™ Red (5 μM in DMSO) and Hoechst 33342 (10 μM in PBS) were added to each well and analyzed at 24, 48, and 72 h. The acquisition parameters were “brightfield” (Channel 1), TexasRed (Channel 2; MitoSox), and DAPI (Channel 3; Hoechst 33342). The “autofocus” was performed in “brightfield,” and the scanned plates with acquisition mode “one field of view per well” with the 4× objective. The images were analyzed using the In-Cell Investigator v1.3 software (GE Healthcare). Mitochondrial superoxide activity was determined based on the “average fluorescence intensity” recorded on the TexasRed channel. Representative DU145 spheroids images were acquired by merging Hoechst (Blue/DAPI) and MitoSox (Red/TexasRed). All analyses were performed with four spheroids/replicates (n = 4) in three biological experiments (n = 3).

All images for the mitochondrial membrane potential (Δψm) analysis were acquired using the HCS platform In-Cell Analyzer 2200 (GE Healthcare) with the MitoStatus Red™ dye (Cat. Nº. 564697; BD Pharmingen™), following the manufacturer’s recommendations. The experimental design and treatments were performed as described for the apoptosis assay reported above. Along with treatments, MitoStatus Red (200 nM in DMSO) and Hoechst 33342 (10 μM in PBS) were added to each well and analyzed at 0.5, 4, 24, 48, and 72 h. The acquisition parameters were “brightfield” (Channel 1), Cy5 (Channel 2; MitoStatus), and DAPI (Channel 3; Hoechst 33342). The “autofocus” was set to “brightfield,” and the plates were scanned with the acquisition mode “one field of view per well” with 4× objective. The acquired images were analyzed using the In-Cell Investigator v1.3 software (GE Healthcare). The Δψm potential was quantified using the “average fluorescence intensity” recorded in the Cy5 channel. Representative images of DU145 spheroids were acquired by merging Hoechst (Blue/DAPI) and MitoStatus (Pink/Cy3). All analyses were performed with four spheroids/replicates (n = 4) and three biological experiments (n = 3).