Cellular Bioluminescence Based Assay: A High Throughput Method for Combinatorial Drug Screening

JoVE Encyclopedia of Experiments

Kankeronderzoek

0 weergaven • 3:17 min • April 30th, 2023

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Cellular bioluminescence is a highly sensitive and rapid approach for large-scale screening of anti-cancer drugs.

This technique uses tumor cells expressing luciferase - an enzyme that catalyzes a light-producing chemical reaction, causing the host cells to glow in the dark. The intensity of the emitted luminescence is directly proportional to the live cell density.

To begin, seed luciferase-expressing cells in an optically clear culture plate coated with an extracellular matrix or ECM and incubate.

During incubation, the ECM promotes the attachment of cells to the culture well.

Next, take concentrated stocks of the drug combination and add different volumes of culture media to prepare serial dilution of the drug mixture.

Now, remove the culture medium from the adhered cells. Add different dilutions of the drug combination and incubate.

During incubation, the drug combination acts synergistically on cellular targets.

Now, remove the drug-containing medium and add fresh medium containing luciferin.

Luciferin diffuses across the membrane and enters the cells. The luciferases expressed by the cells use cellular ATP to oxidize luciferin and emit light signals.

Quantify the cellular bioluminescence under an imaging system.

A decrease in light emission with increased drug concentration indicates reduced cell viability. This correlates to the drug's antiproliferative effect on the target cells.

Coat 96-well optical bottom plates with an extracellular matrix mixture such as Matrigel and incubate them for 1 hour at 37 degrees Celsius. Remove the excess mixture and gently rinse the plates once with PBS.

Then, seed XG387-Luc cells at a density of 1,000 cells in 100 microliters of culture medium into each well of the coated plates and culture them overnight. On the following day, observe the cells under a microscope to confirm their attachment to the plate.

Next, prepare a 200-micromolar temozolomide solution and 2-micromolar solutions of the targeted agents in culture medium. For combination drug screening, remove the blank medium and add the medium containing either 200-micromolar temozolomide or 2-micromolar targeted agent, or a combination of both, into each well for 3 technical replicates per treatment.

Incubate the plate at 37 degrees Celsius and 5% carbon dioxide for 3 days. Take images of the cellular bioluminescence in the plate using the IVIS spectrum imaging system. Using the built-in software, create multiple circular areas of the region of interest and quantify the cellular bioluminescence.

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Laatst bijgewerkt: 15 augustus 2026