Bioluminescence is a powerful tool for monitoring diverse biological functions within cells. Traditional bioluminescent assays use firefly (luc) or Renilla (Rluc) luciferases. They require the addition of activating chemical luciferin and destruction of cellular samples. Thus, the system produces intermittent data snapshots that cannot accurately represent cellular behavior. However, bacterial luciferase is the only known bioluminescent system with a genetically-defined luciferase/luciferin synthesis pathway functional at the mammalian cell temperature optimum of 37 °C1. Here, we describe how mammalian cells expressing bacterial luciferase can transition endpoint luciferase assays to continuous, kinetic formats that produce more information from fewer samples.
Kinetic measurement of mammalian cellular viability and metabolic dynamics in response to drug treatment is fundamental for the progression of biomedical research. In this example, we validate the use of endogenous bioluminescent production to track viability in response to the potent antibiotic Zeocin. We then show that continuous monitoring can reveal dynamic metabolic effects by treating with the anti-tumor pharmaceutical ellipticine. Ellipticine was chosen because, like Zeocin, it intercalates DNA. At high doses it inhibits electron transfer in mitochondria and impacts cellular viability by depleting ATP. However, lower doses uncouple oxidative phosphorylation and increases metabolic activity2.
Bioluminescent assays measure metabolic activity by detecting the presence of specific metabolites within the cell. For example, luc assays measure ATP content, with higher ATP levels representing increased metabolic activity. Bacterial luciferase assays work similarly, with FMNH2 serving as the measured metabolite. Greater FMNH2 availability increases light output, representing increased metabolic activity3. 490 BioTech offers commercially available autobioluminescent cells that express all the genes required for both bacterial luciferase and luciferin synthesis pathways. Because both components are endogenously available, light is produced continuously while signal intensity is constitutively modulated in response to FMNH2 availability. This allows non-stop tracking of metabolic activity in real-time via measurement of light output. By comparing signal intensity to the pre-treatment baseline, we observed positive, negative, or transient effects on cellular function as they occurred.
Materials & Methods
- White, 96-well plates (Corning)
- CLARIOstar with ACU (BMG LABTECH)
- Zeocin (Thermo Fisher Scientific)
- Ellipticine (ApexBio)
- Autobioluminescent HEK293 cells (490 BioTech)
Autobioluminescent HEK293 cells were seeded in triplicate at 10,000 cells/well and allowed to adhere at 37 °C for 24 hours in DMEM media containing 10% FBS. Cells were challenged with increasing concentrations of Zeocin (200-1,000 µg/mL) or ellipticine (500 nM and 5 µM). Vehicle controls were included to establish baseline metabolic activity. The microplate was immediately placed into the CLARIOstar microplate reader and adjusted to the atmospheric conditions set by the ACU for 4 hours. Bioluminescent output was monitored for two days to track metabolic activity and viability.
||Luminescence, plate mode, no filter|
|Focal height||11.0 mm|
||Interval time||10 s|
|Settling time||0 s|
|Kinetic settings||Number of cycles||97|
|Cycle time||1800 s|
|Incubation||30 °C, 5% CO|
Results & Discussion
The CLARIOstar with ACU, combined with self-modulating autobioluminescent cells, permits continuous monitoring without perturbation for long time periods. Figure 2 illustrates that Zeocin reduces cellular viability in a dose-dependent fashion as expected. Autobioluminescent output is decreased at each treatment level, with decreasing viability observed at higher dosages. Cells exposed to 1,000 µg/mL were completely non-viable by 24 hours post treatment, as indicated by cessation of autobioluminescence. 200 µg/mL Zeocin treatment depressed metabolic activity, as indicated by an initial decrease in autobioluminescence, but effected cells either recovered or were replaced by the doubling of non-effected cells as autobioluminescence increased beyond the 24 hour time point.
Autobioluminescent cells should be capable of revealing positive or transient metabolic effects as well. To demonstrate these effects, we challenged samples with the anti-tumor pharmaceutical ellipticine (Figure 3). As expected, metabolic activity increased at low dosage (500 nM). High dosage (5 µM) initially depressed metabolic activity. Over time, the supplied ellipticine was metabolized, bioavailability decreased, and the effect of the drug transitioned from negative to positive. Because metabolism could be monitored continuously, it was possible to observe the timing and duration of these effects from a single sample set without perturbation.Conclusion
Assessment of cellular responses to drug treatment can be performed in real-time by measuring autobioluminescent output in cultured human cells. This is made possible by the CLARIOstar microplate reader with atmospheric control unit to provide cell culture conditions. Thus, autobioluminescent cells allow continuous monitoring of both cellular viability and drug metabolism. This increased informational capacity can be used to inform biomedical and drug development research.
- Szittner R et al. Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium. J. Biol. Chem. 265, 16581-16587 (1990).
- Schwaller MA et al. Protonophoric activity of ellipticine and isomers across the energy-transducing membrane of mitochondria. J. Biol. Chem. 270, 22709-22713 (1995).
- Close, D. M. et al. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line. PLoS ONE 5, e12441 (2010).