Real-Time Monitoring Of Cell Health In 3D Cell Cultures

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 My name is Darren Heywood, and I'm one of the product managers at Promega. Today, I'll give you a brief overview of our real-time kinetic cell-based assays for monitoring cell health in 3D cell cultures.

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 Real-time assays enable you to repeatedly measure specific events or conditions over time from the same plate, either from cells or culture media.

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 Our assays tend to have small molecule probes, which can be a pro-substrate, a DNA-binding dye, or an analyte secreted into the media. You can sample the media and build up a real-time picture of what's happening in the cell.

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 Real-time assays allow you to collect data without lysing the cells, which means you can multiplex several assays within your experiment.

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 Multiplexing different assays allows you to gain more data from your sample. For example, you can use a viability assay to normalize against the number of cells in your culture.

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 Using a single plate means you'll use fewer reagents, cells, and culture media which saves resources. Additionally, because you're not lysing the cells, you can use them for downstream processing.

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 Promega has many validated real-time 3D cell-based assays for continuously monitoring viability, cytotoxicity, mechanisms of death, and metabolism. These assays can measure in real-time for hours or even days.

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 Our assays are homogeneous, meaning they use add-mix-measure methods, making them easy to use and scalable for high-throughput applications.

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 These are the assays we have for 3D cell culture. Most are endpoint assays, but the ones highlighted in orange are real-time assays, which I'll discuss today.

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 When you add a compound to your cells, you might want to know how it affects viability. We have a real-time assay called the RealTime-Glo™ MT Cell Viability Assay, a luminescent assay used to monitor viability over time.

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 The RealTime-Glo™ assay is used to measure cell viability. It consists of two components: a pro-substrate and NanoLuc® luciferase. These are added to your cells when you seed or dose them with your compound.

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 The cell-permeable pro-substrate gets into the cell, is reduced by intracellular reduction, diffuses out, and binds to luciferase, generating light proportional to the number of viable cells in your culture.

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 This doesn't happen in dead cells because they aren't metabolically active and can't reduce the pro-substrate.

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 Here's an example where we've used this assay. We treated hepatocytes with increasing concentrations of Panobinostat, resulting in a dose-dependent decrease in viable cells over time.

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 Typically, you can read out viability for up to three days, but by spiking in more reagent during media changes, you can extend this three day period.

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 Now, let's look at cell death, does your compound effect cell death? We have two assays for this: CellTox™ Green and LDH-Glo™. CellTox™ Green is a real-time fluorescent assay, while LDH-Glo™ is a luminescent assay. Both rely on the breakdown of the membrane during cell death.

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 CellTox™ Green is a cell-impermeable asymmetric cyanine dye added to a buffer and then to your cells. It binds to DNA in dead cells, enhancing its fluorescent properties.

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 This doesn't happen in viable cells because the membrane is intact, preventing the dye from entering.

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 Here's an example where we've used this assay. We treated HepG2 spheroids with paclitaxel in the presence of CellTox™ Green, taking images from 0 to 48 hours, showing a progression of cell death. This is shown by the fluorescence

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 We multiplexed this assay with CellTiter-Glo® 3D, a lytic viability assay that lyses cells, releasing ATP used in the luciferin-luciferase reaction to generate light.

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 Paclitaxel resulted in a dose-dependent increase in dead cells and a decrease in viable cells.

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 When treating mixed populations of cells, they don't all die at the same rate. You can remove those populations and use them for downstream processing, like DNA and RNA extraction.

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 Here's how it looks: at 0 hours, no dead cells; at 22 hours, some cells start to fluoresce; at 48 hours, the whole plate glows. You can remove individual wells for transcriptomics and continue incubating the plate.

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 The LDH-Glo™ assay measures lactate dehydrogenase released when a cell dies. You sample the media and add LDH detection reagent, producing luciferin and generating light proportional to the number of dead cells.

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 Here's an example where we've used this assay. We treated HCT116 spheroids with doxorubicin, sampling the media at 24, 48, and 72 hours post-treatment. Doxorubicin resulted in a dose-dependent increase in dead cells and a decrease in viable cells.

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 You can multiplex the RealTime-Glo™ and CellTox™ Green assays to look at viability and cytotoxicity from the same sample in real-time.

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 We've looked at viability and cytotoxicity in real-time, but what about the mechanism of cell death? We have two real-time assays for this: RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay and RealTime-Glo™ Extracellular ATP Assay.

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 The RealTime-Glo™ Annexin V assay uses NanoLuc® luciferase, split into a large and small subunit with low affinity for each other. We've conjugated Annexin V to these subunits and included a cell-impermeable necrosis detection reagent.

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 During early apoptosis, phosphatidylserine flips from the inside to the outside of the membrane. Annexin V binds to it, bringing together the luciferase subunits and generating light. The necrosis detection reagent can't enter the cell at this stage.

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 As the cell progresses to necrosis, the membrane breaks down, allowing the necrosis detection reagent to enter and generate fluorescence.

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 Here's an example where we've used this assay. We treated HepG2 spheroids with paclitaxel for 48 hours. At 6 hours, not much happens; at 22 hours, apoptosis begins; at 30 hours, cells progress to secondary necrosis; at 48 hours, cells are in secondary necrosis.

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 The RealTime-Glo™ Extracellular ATP Assay measures immunogenic cell death by detecting ATP released during cell death. The amount of light generated is proportional to the amount of immunogenic cell death.

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 Here's an example where we've used this assay. We treated HCT116 spheroids with staurosporine, resulting in a dose-dependent increase in extracellular ATP and immunogenic cell death.

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 We also have assays for energy metabolism: Glutamine-Glo™, Glutamate-Glo™, Glucose-Glo™, and Lactate-Glo™. These luminescent assays measure specific metabolites in the media.

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In summary, real-time assays provide a complete picture of cellular changes, allowing you to multiplex different assays and explore multiple aspects of your model system. They offer status updates during experiments, unveiling trends that endpoint assays might miss.

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 For more information, visit our website or join our LinkedIn groups. If you have any questions, please see us at the stand or drop me an email.