Cell event green

Chip replicates designated for live cell imaging were washed with PBS utilizing the bubble method. Chips were then cut in half perpendicular to the co-culture channels. The top chip halves were stained with NucBlue (ThermoFisher, R37605) to visualize cell nuclei and Cell Event Green (ThermoFisher, C10423) to visualize activated caspase 3/7 for apoptosis. This staining panel was prepared in serum-free media (CSC), with NucBlue at 2 drops per mL and Cell Event Green at a 1:500 ratio and perfused through both channels. The bottom chips halves were stained with NucBlue (Thermo) to visualize nuclei and Tetramethylrhodamine, methyl ester (TMRM) (ThermoFisher, I34361) to visualize active mitochondria. This staining panel was prepared in PBS with 5% FBS, with NucBlue at 2 drops per mL and TMRM at a 1:1000 ratio in original blocking buffer, diluted 1:4 in PBS. Chips were incubated in the dark at 37 °C for 30 min, and then each channel was washed with 200 µL of PBS (alternating channels, 2× for top and 3× for bottom). The chips were kept at 37 °C, protected from light, until ready for imaging with the Opera Phenix.

Cell Event Green (ThermoFisher, C10423) and SytoxRed (ThermoFisher, S11380) were used to assess the modes (apoptosis and necrosis, respectively) and timing of cell death at 4 hours, 1, 3 and 5 days post-freeze. Cell Event Green (caspase 3/7 activation) was used to assess apoptotic involvement whereas Sytox Red indicated necrosis. Samples were incubated for 30 minutes at 37°C with 5 μM Cell Event in 1X PBS, followed by a 15 minute 37°C incubation with 500 nM Sytox Red. Samples were counterstained with 3 μg/mL Hoechst 33342 (Invitrogen, H3570) in 1X PBS and then washed for 5 minutes in 1X PBS. Samples were then analyzed with the Cell Insight CX5 high content screening platform (ThermoFisher) using the Target Activation Assay protocol. A minimum of 3 experimental repeats with an intra experimental repeat of 3 wells was performed in each condition (n ≥ 9). Statistical significance was determined by single factor ANOVA.