Introduction
Analyses of cell viability and cytotoxicity are examples of cell-based assays that derive great benefit from the ability to detect changes to these parameters in real time. Real-time detection allows identification of an exact moment in time when a cytotoxic or antiproliferative change occurs. When these assessments are performed with a microplate reader multiple samples and concentrations can be monitored on a single plate.
Two assays from Promega (RealTime-Glo® MT Cell Viability and the CellTox Green Cytotoxicity Assay) were measured over 72 hours. In order to maintain cell health the CLARIOstar was equipped with an Atmospheric Control Unit (ACU). This enables the detection of both time- and dose-dependent effects on cell proliferation and cytotoxicity.
Assay Principle
The RealTime-Glo® MT Cell Viability Assay is a bioluminescent assay that relies on the metabolic (MT) reducing potential of cells. NanoLuc® luciferase and cell-permeant pro-NanoLuc® substrate are added to cells in culture. Viable cells reduce the substrate which then diffuses into the medium where it is rapidly used by NanoLuc® enzyme to produce a luminescent signal proportional to viable cell number (Fig. 1).
The non-activity based CellTox™ Green Cytotoxicity Assay is comprised of a cell membrane impermeant dye that is excluded from viable cells. When the cell membrane becomes compromised, the dye enters the cell where it binds to DNA and becomes fluorescent. Fluorescent signal is proportional to the number of dead cells in culture (Fig. 2).
Materials & Methods
- 384-well, white, clear bottom microplates from Corning
- RealTime-GloTM MT Cell Viability Assay from Promega
- CellToxTM Green Cytotoxicity Assay from Promega
- CLARIOstar® microplate reader from BMG LABTECH
Fluorescence instrument settings
| Method: | Bottom reading |
| Flashes per well: | 50 |
| Optic Settings: | Excitation: F: 482-16/ Emission F: 530-40 |
| Dichroic: | LP 504 |
| No. of cycles: | 73 |
| Cycle time: | 60 min |
| Gain + Focus: | adjusted prior to test |
Luminescence instrument settings
| Measurement interval time: | 1.0 s |
| Optic settings: | No Filter |
| No. of cycles: | 73 |
| Cycle time: | 60 min |
| Gain: | 3500 |
| Cycle time: | 60 min |
| Gain + Focus: | adjusted prior to test |
ACU settings
| CO2: | 5 % |
| O2: | monitoring |
| Target temperature: | 37°C |
Results & Discussion
The CLARIOstar with ACU was able to fully sustain the normal proliferation and health of untreated cells for the entire 72 hour time course (Fig. 3).
Cells treated with varying concentrations of the tyrosine kinase inhibitor bosutinib, initially exhibit proliferation although higher concentrations suppressed proliferation. For all but the lowest concentration, a change appears to occur around 25 hours and cell viability begins to decrease (Fig. 4).
All concentrations of bosutinib also increased cytotoxicity to some degree. Cytotoxicity begins to increase at around 30 hours coincident with decreased viability (Fig. 5).
Conclusion
The CLARIOstar with ACU keeps cells happy so that long term cell-based assays can be achieved. In this case both cell viability and cytoxicity were monitored over 72 hours. Here we show the hourly assessment of the effect of the tyrosine kinase inhibitor bosutinib. The results show a clear time dependence for cytotoxicity as well as time and dose dependence for cell viability.