2.5. MitoSox red staining

We recommend optimizing the procedure for dissociated mouse hepatocyte spheroids, as the dissociation process itself may affect mitochondrial ROS levels and cell viability.

• MitoSOX stock preparation and working concentration
  MitoSOX Red (Thermo Fisher Scientific) was dissolved in DMSO to prepare a 5 mM stock solution. Immediately before use, the stock was diluted in pre-warmed DPBS to a final working concentration of 5 μM. Stock aliquots were protected from light and repeated freeze–thaw cycles were avoided. 

• Cell number and staining volume used per sample
  In our study using mouse embryonic stem cells, we typically stained approximately 1–2 × 10⁵ cells per sample in 200 μL of pre-warmed DPBS containing 5 μM MitoSOX Red. Cell density and staining volume were kept consistent across experimental groups. 

• Buffer used during incubation
  Cells were stained in pre-warmed DPBS. 

• Incubation conditions
  Cells were incubated with 5 μM MitoSOX Red for 10 min at 37°C in the dark.

• Washing steps
  Following staining, cells were washed three times with pre-warmed DPBS and pelleted after each wash for 1 min. 

• Staining format
  Cells were maintained in suspension during staining. 

• Timing of flow-cytometric acquisition
  Samples were analyzed immediately after the final wash and, whenever possible, within 30 min after completion of staining. 

• Flow-cytometer configuration
  Samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences), with 488-nm excitation and the FL2 channel equipped with a 585/42-nm band-pass filter. Instrument voltage and gating settings should be optimized using appropriate controls on the individual instrument. 

• Quantification
  MitoSOX fluorescence was quantified as the geometric mean fluorescence intensity of the gated cell population. Median fluorescence intensity may also be used, provided that the same metric and gating strategy are consistently applied across all samples. 

• Gating and viability exclusion
  Debris was excluded based on FSC/SSC characteristics. A dedicated viability dye was not included in our original analysis. For dissociated hepatocyte spheroids, we recommend including a compatible live/dead discriminator, as dead or damaged cells can exhibit nonspecific fluorescence. Where available, doublets may be excluded using FSC-area versus FSC-height or FSC-width. 

• Controls for mitochondrial ROS
  We did not include pharmacological positive or negative controls for mitochondrial ROS in the original study. For assay optimization in dissociated hepatocyte spheroids, however, published studies commonly use mitochondrial electron transport chain inhibitors as positive controls. For example, antimycin A (typically 1–10 μM), which inhibits complex III, can increase mitochondrial superoxide production. Rotenone (typically 1–50 μM for 10–30 min, depending on the cell type), which inhibits complex I, may also be used to increase mitochondrial ROS.

These concentrations and treatment durations should be empirically optimized for the relevant cell type and interpreted together with cell-viability measurements, as both compounds can substantially affect mitochondrial function and cell survival. An unstained control should be included to establish background fluorescence.