Table 1 Summary of marker optimizations and results
Marker Type | Marker Assay | Optimal incubation time | S:B ratio ≥ 3:1? | Correlation to NTS viability/cytotoxicity | Selected for drug assay testing? | Justification |
|---|---|---|---|---|---|---|
Viability Markers | CellTiter-Glo® | 15 min | Yes, with ≥ 100 NTS | Strong | Yes | Met criteria |
Resazurin | 24 hours | Yes, with ≥ 200 NTS | Strong | Yes | Met criteria | |
Cytotoxicity Markers | Vybrant® | 70 min | Almost, with ≥ 300 NTS | Strong up to 300 NTS | Yes | Met criteria |
CytoTox-ONE | 2 hours | No | Strong | No | S:B ratio low, large standard deviations between data points | |
CellTox™ Green Cytotoxicity Assay | 24 hours | Yes, with ≥ 400 NTS | Strong with ≥ 400 NTS | No | Viable NTS died within 24 hours of exposure to dye; strong signals from completely lysed cells only | |
Multiplex Markers | LIVE/DEAD® Viability/Cytotoxicity Kit | Does not exist | No | Poor for both live and dead NTS | No | Poor correlation to NTS viability/cytotoxicity |
ApoTox-Glo™ | Does not exist | No | Strong for live-cell marker, poor for dead-cell marker | No | Markers induced spazzing and death of NTS after 6 hours | |
Experimental Markers | OmniCathepsin™ | 2 hours (with 10 μM marker concentration) | No | Good: Differential signals for live vs. dead observed | No | S:B ratio too low, not enough difference between live and dead NTS |
FluoForte® Calcium Assay | 1.5 hours with ≥ 200 dead NTS | Yes, with ≥ 200 dead NTS (lysed only) | Poor: strong staining for completely lysed cells only | No | Stains only completely lysed cells | |
DAPI | 15 min (with 10 μg/ml dye concentration) | No | Good: stained many dead NTS phenotypes | No | Background fluorescence too high, signals too low | |
Hoechst 33258 | 15 min (with 1 μg/ml dye concentration) | No | Good: stained many dead NTS phenotypes | No | Background fluorescence too high, signals too low |