Reveal More Biology: How Real-Time Kinetic Cell Health Assays Prove Their Worth

What if you could uncover a small but significant cellular response as your population of cells move toward apoptosis or necrosis? What if you could view the full picture of cellular changes rather than a single snapshot at one point? You can! There are real-time assays that can look at the kinetics of changes in cell viability, apoptosis, necrosis and cytotoxicity—all in a plate-based format. Seeking more information? Multiplex a real-time assay with endpoint analysis. From molecular profiling to complementary assays (e.g., an endpoint cell viability assay paired with a real-time apoptosis assay), you can discover more information hidden in the same cells during the same experiment.

Whether your research involves screening a panel of compounds or perturbing a regulatory pathway, a more complete picture of cellular changes gives you the benefit of more data points for better decision making. Rather than assessing the results of your experiment using a single time point, such as 48 hours, you could monitor cellular changes at regular intervals. For instance, a nonlytic live-cell reagent can be added to cultured cells and measurements taken repeatedly over time. Pairing a real-time cell health reagent with a detection instrument that can maintain the cells at the correct temperature means you can automate the measurements. These repeated measurements over time reveal the kinetic changes in the cells you are testing, giving a real-time status update of the cellular changes from the beginning to the end of your experiment. Continue reading

Piecing the Puzzle Together: Using Multiple Assays to Better Understand What Is Happening with Your Cells

You often need several pieces of information to really understand what is happening within a cell or population of cells. If your cells are not proliferating, are they dying? Or, are you seeing cytostasis? If they are dying, what is the mechanism? Is it apoptosis or necrosis? If you are seeing apoptosis, what is the pathway: intrinsic or extrinsic?

If you are measuring expression of a reporter gene and you see a decrease in expression, is that decrease due to transfection inefficiencies, cytotoxicity, or true down regulation of your reporter gene?

To investigate these multiple parameters, you can run assays in parallel, but that requires more sample, and sample isn’t always abundant.

Multiplexing assays allows you to obtain information about multiple parameters or events (e.g., reporter gene expression and cell viability; caspase-3 activity and cell viability) from a single sample. Multiplexing saves sample, saves time and gives you a more complete picture of the biology that is happening with your experimental sample.

What information do you need about your cells to complete the picture?

What information do you need about your cells to complete the picture?

Multiplexing assay reagents to measure biomarkers in the same sample has often been considered an application only accomplished with antibodies or dyes and sophisticated detection instrumentation. However, Promega has developed microwell plate based assays for cells in culture that allow multiplexed detection of biomarkers in the same sample well using standard multimode multiwell plate readers. Continue reading

The 64 billion dollar question: Is my compound or treatment toxic?

CellTox™ Green Dye is excluded from viable cells, but it binds to DNA from cells with compromised membrane integrity.

CellTox™ Green Dye is excluded from viable cells, but it binds to DNA from cells with compromised membrane integrity.

Determining the exact cause/effect relationship between a treatment and a cellular outcome is not a simple matter, but is critical for really understanding how therapeutic treatments affect target cells or exercise any off-target effects.

Four key factors are critical for determining whether or not a particular treatment or compound is toxic.

  1. Dosage (usually addressed by a dilution series)
  2. Exposure time
  3. Mechanism of Action
  4. Cell Type

In a recent Promega Webinar, A Cytotoxicity Assay That Fits Your Timeline, Promega scientist Dr. Andrew Niles presented the CellTox™ Green Cytotoxicity Assay—a new tool that gives researchers more power to answer the question “Is my compound or treatment toxic?” Continue reading

Black Raspberry Extract May Lead to Tomorrow’s Cancer Preventative

Black raspberry shrubs. Copyright Sara KlinkWhen deciding which varieties of fruit to cultivate, I chose to plant black raspberries on my small suburban lot. They grow wild in Wisconsin, but fighting through swarms of mosquitos, brush and thorns to pick berries was not my idea of fun. For the last two years, I have received a large crop of juicy black berries that I enjoy eating fresh or process into black raspberry jam to spread on toast. Therefore, I was interested to learn that black raspberries have demonstrated cancer preventative properties in animal models of chemically induced oral and colon cancers as well as cultured oral cancer cells. Due to similarities between oral and cervical cancers, researchers recently tested if the beneficial effects of this berry could extend to human cervical cancer cells. Continue reading

Screening for Drug-Drug Interactions with PXR and CYP3A4 Activation

Cytochrome P450 3A4 Enzyme

Numerous pharmaceutical companies have adopted assays for detecting activation of pregnane X receptor (PXR), a nuclear receptor known to regulate expression of cytochrome P450 (CYP450) drug-metabolizing enzymes (1). PXR is a transcription factor that has been designated the “master xenosensor” due to its ability to upregulate cellular levels of a variety of drug-metabolizing enzymes in response to drugs and foreign chemicals. Elevated levels of CYP450 enzymes can elicit alterations in the pharmacokinetics of co-administered drugs, which can result in adverse drug-drug interactions (DDI) or diminished bioavailability. By assessing PXR activation and CYP450 enzyme induction early in the drug development process, many companies hope to reduce late-stage clinical failures and minimize the high costs associated with bringing a new drug to market.

Proportion of drugs metabolized by different CYPs

A recent paper by Shukla et al. (2) examined over 2,800 clinically used drugs for their ability to activate human PXR (hPXR) and rat PXR (rPXR), induce human cytochrome P450 3A4 enzyme (CYP3A4) at the cellular level, and bind hPXR at the protein level. Several studies have identified PXR as playing a key role in regulating the expression of CYP3A4, an enzyme involved in the metabolism of more than 50% of all drugs prescribed in humans. Continue reading

Predictive In Vitro Cytotoxicity Testing

Cell culture cytotoxicity testing is used as a predictor for animal toxicity. High-throughput cytotoxicity screening using ATP levels as an indicator of cell viability is the current gold standard for such predictive cytotoxicity testing. Multiplexing assay chemistries allows researchers to measure multiple parameters on a single sample in order to get a more complete picture of what is happening when cells are exposed to a treatment compound. For example multiplex assays using three protease activities as markers of viable, necrotic and apoptotic cells give researchers a tool for uncovering the mechanism of cell death when toxicity is observed and control for assay artifacts. In their book chapter, “Cytotoxicity Testing: Measuring Viable Cells, Dead Cells and Detecting Mechanism of Cell Death”, Riss, Moravec and Niles, describe protocols for in vitro toxicity testing using ATP-based assays and multiplex assays. The chapter provides protocols, an extensive materials required list, example data, and a thorough notes section describing appropriate controls, issues of assay timing, and other considerations that affect assay success. You can find it in Methods in Molecular Biology Vol. 740, Mammalian Cell Viability Methods and Protocols (Humana Press).

Describing Life and Death in the Cell

4621CALife is complicated. So is death. And when the cells in your multiwell plate die after compound treatment, it’s not enough to know that they died. You need to know how they died: apoptosis or necrosis? Or, have you really just reduced viability, rather than induced death? Is the cytotoxicity you see dose-dependent? If you look earlier during drug treatment of your cells, do you see markers of apoptosis? If you wait longer, do you observe necrosis? If you reduce the dosage of your test compound, is it still cytotoxic? Continue reading