In the Literature

Blogs about published peer-reviewed scientific studies.

A Cell Viability Assay for Today

Posted on by Isobel Maciver
Valued for ease of use and scalability, plate-based, bioluminescent cell viability assays are widely used to support research in biologics, oncology and drug discovery.

Cell viability assays are a bread-and-butter method for many researchers using cultured cells —everyday lab tools that are a part of many newsworthy papers, but rarely make news themselves.

Over time, cell viability assays have become easier to use and more “plug ‘n play”. Among modern assays, luminescent plate-reader based systems have been a favorite for several years because of their superior sensitivity, robustness, simple protocols and uncomplicated equipment requirements (all you need is a plate-reading luminometer). These qualities combine to allow easy scalability and adaptability from bench research to high throughput applications.

CellTiter-Glo® Luminescent Cell Viability Assay is an accepted go-to viability assay for many researchers. The assay measures ATP as an indicator of metabolically active cells. A quick search on Google Scholar returns 3,990 CellTiter-Glo results for 2017 and over 500 so far in January and February of 2018. A sampling of these recent publications gives a snapshot of some of the ways the CellTiter-Glo assay is used to support key areas of research today.

Does a treatment kill cells?

The obvious application of a cell viability assay is to understand whether cells are alive. In cancer research, the CellTiter-Glo assay is often used to confirm killing of tumor cells and to verify that normal cells survive. Therefore, these assays are a key part of the evaluation and screening of drug candidates and other therapies for cancer. Many papers reporting use of CellTiter-Glo are developing and evaluating the effectiveness of novel anti-cancer treatments. Continue reading “A Cell Viability Assay for Today”

It’s a Girl! Welcoming Black-Footed Ferret Kit Elizabeth Ann!!

Posted on by Kari Kenefick

Updated February 2021.

In February 2018 we wrote about a resurrection effort to bring the then endangered black-footed ferret back from the brink of extinction in western U.S. This effort was undertaken by the U.S. Fish and Wildlife Service, with assistance from Revive & Restore and partners ViaGen Pets & Equine, San Diego Zoo Global and the Association of Zoos and Aquariums.

On February 18, the U.S. Fish and Wildlife Service announced announced the successful cloning of a black-footed ferret, introducing the world to a 38-day-old black-footed ferret kit “Elizabeth Ann” cloned from cells of a female ferret that died in 1988.

Photos of Elizabeth Ann, the first cloned black footed ferret. Image credit: US Fish and Wildlife Service

Cells from ferret, “Willa” were preserved by freezing, and when somatic cell nuclear transfer (SCNT) became a possibility, Willa’s cells were used to create Elizabeth Ann, the kit born just over one month ago.

Before Elizabeth Ann’s birth there were upwards of 1,000 black-footed ferrets alive in the western U.S., but they were all descendants of just 7 ferrets, and thus genetically very similar.

Analysis of Elizabeth Ann’s genome has revealed more than three times the genetic variants found in the existing wild U.S. ferrets. This means that if she is able to reproduce, her contribution to the genetic diversity of wild ferrets would be huge.

Interested in learning more about ferrets and the challenges they’ve faced in surviving and thriving in the wild? Below is our original 2018 blog with those details. Don’t miss the video clip of a young black-footed ferret doing the “weasel war dance” (below).

Continue reading “It’s a Girl! Welcoming Black-Footed Ferret Kit Elizabeth Ann!!”

Mass Spec Analysis of PTMs Using Minimal Sample Material

Posted on by Gary Kobs

DNA is organized by protein:DNA complexes called nucleosomes in eukaryotes. Nucleosomes are composed of 147 base pairs of DNA wrapped around a histone octamer containing two copies of each core histone protein. Histone proteins play significant roles in many nuclear processes including transcription, DNA damage repair and heterochromatin formation. Histone proteins are extensively and dynamically post-translationally modified, and these post-translational modifications (PTMs) are thought to comprise a specific combinatorial PTM profile of a histone that dictates its specific function.  Abnormal regulations of PTM may lead to developmental disorders and disease development such as cancer.

Continue reading “Mass Spec Analysis of PTMs Using Minimal Sample Material”

A Virus-like Neural Pathway Hints at the Origins of the Mammalian Brain

Posted on by Johanna Lee

The mammalian brain is extremely complex. We know that it processes and stores information through synaptic connections within a complicated neural network. But how exactly do neurons communicate with each other? And how did this neural network come to exist? A recent paper published in Cell may provide some answers. It describes a previously unknown signaling pathway–with surprising origins–that transports RNA between neurons. Continue reading “A Virus-like Neural Pathway Hints at the Origins of the Mammalian Brain”

Two Epigenetic Targets Are More Effective Than One

Posted on by Sara Klink

Lysine-specific histone demethylase 1 (LSD1) via Wikimedia Commons

Epigenetics is a new and exciting territory to explore as we understand more about the role it plays in gene silencing and expression. Because epigenetic regulation of gene expression is caused by specific modification of histone proteins (e.g., methylation) that play a role in disease states like cancer, enzymes like histone deacetylases (HDACs) become viable drug targets. One drawback to inhibiting proteins that modify histones is even when selectively targeting HDACs, the effects can be far ranging with multiple HDAC-containing protein complexes found throughout the cell. These broad effects minimize the effectiveness of an inhibitor, caught between efficacy and toxicity. A recent article in Nature Communications explored how using a single compound to target two epigenetic enzymes was more effective than any individual inhibitor or combination of inhibitors. Continue reading “Two Epigenetic Targets Are More Effective Than One”

Luciferase Immunoprecipitation System Assay (LIPS): Expression of Luciferase Antigen using TNT Transcription/Translation Kit

Posted on by Gary Kobs

NanoLuc dual reporters
Illustration showing NanoLuc and firefly luciferase reporters.

The luciferase immunoprecipitation system (LIPS) assay is a liquid phase immunoassay allowing high-throughput serological screening of antigen-specific antibodies. The immunoassay involves quantitating serum antibodies by measuring luminescence emitted by the reporter enzyme Renilla luciferase (Rluc) fused to an antigen of interest. The Rluc-antigen fusion protein is recognized by antigen-specific antibodies, and antigen-antibody complexes are captured by protein A/G beads that recognize the Fc region of the IgG antibody (1).

In a recent publication (2), this assay was used to assess the presence of autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in patients with atrophic body gastritis and in controls. Continue reading “Luciferase Immunoprecipitation System Assay (LIPS): Expression of Luciferase Antigen using TNT Transcription/Translation Kit”

Ancient Images of Dogs Include Restraints?

Posted on by Kari Kenefick
This dog is wearing a leash.

You, like me, may occasionally find youself in need of a canine control device. While I’m not a fan of the dog tie out, I do walk dogs on leash—as is required by our county and city government here in Madison, WI.

If you have read Temple Grandin’s books about dogs, you might feel a tug at your heartstrings while enduring a tug on the leash. Aren’t dogs meant to run freely? Don’t we love to watch them run? Are leashes humane?

When walking dogs I feel the need to protect them, but also a desire to let them live like dogs, sniffing, marking and other behaviors that are all limited when the dog is leashed.

When a report in Science last week showed evidence that our ancient ancestors were using leashes 8,000-9,000 years ago I was: 1) surprised; and 2) felt vindicated from self-imposed dog owner guilt.

Continue reading “Ancient Images of Dogs Include Restraints?”

Using CellTiter-Glo® Luminescent Cell Viability Assay to Assess Cell Viability in Cancer Cells Treated with Silver Nanoparticles and DNA-PKcs Inhibitor

Posted on by Johanna Lee

Silver nanoparticles (Ag-np) are commonly used in many consumer products, including cosmetics, textiles, electronics and medicine, largely due to their antimicrobial properties. More recently, Ag-np are being used to target and kill cancer cells. It has been known for years that silver nanoparticles (Ag-np) can induce cell death and DNA damage. Studies have also shown that Ag-np inhibit cell proliferation and induce apoptosis in cancer cells. However, cancer cells are able to fight back with DNA repair mechanisms such as non-homologous end joining repair (NHEJ). The NHEJ pathway requires the activation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), thus DNA-PKcs may protect against the Ag-np-induced DNA damage in cancer cells.

Could inhibition of DNA-PKcs increase the ability of Ag-np to kill cancer cells? In a 2017 study, Lim et al. wanted to test whether inhibition of DNA-PKcs can increase the cytotoxic effect of Ag-np in breast cancer and glioblastoma cell lines. To effectively determine cell viability in these cancer cell lines, the authors used the CellTiter-Glo® Luminescent Cell Viability Assay. The CellTiter-Glo® Assay determines the number of viable cells in culture based on quantitation of ATP, an indicator of metabolically active cells. A major advantage of this assay is its simplicity. This plate-based assay involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. This generates a luminescent signal proportional to the amount of ATP present, which is detected using a luminometer. Cell washing, removal of medium and multiple pipetting steps are not required. Another advantage of the CellTiter-Glo® Assay is its high sensitivity. The system detects as few as 15 cells/well in a 384-well format in 10 minutes after adding reagent and mixing, making it ideal for automated high-throughput screening, cell proliferation and cytotoxicity assays.

The authors first confirmed that Ag-np treatment reduced proliferation and induced cell death/DNA damage in two breast cancer cell lines and two glioblastoma cell lines. The cytotoxic effect of Ag-np is specific to cancer cells, as minimal cytotoxicity was observed in non-cancerous human lung fibroblasts used as control. Next, they pre-treated the cancer cells with a DNA-PKcs inhibitor for 1 hour before adding Ag-np. Inhibition of DNA-PKcs increased Ag-np-mediated cell death in all four cancer cell lines. This suggests that DNA-PKcs may be protecting the cells from Ag-np cytotoxicity. The authors further showed that DNA-PKcs may repair Ag-np induced DNA damage by NHEJ and JNK1 pathways. In addition, DNA-PKcs may help recruit DNA repair machinery to damaged telomeres.

This study suggests that a combination of Ag-np treatment and DNA-PKcs inhibition may be a potential strategy to enhance the anticancer effect of Ag-np.

Reference: Hande M.P., et.al. (2017) DNA-dependent protein kinase modulates the anti-cancer properties of silver nanoparticles in human cancer cells. Mutat Res Gen Tox En. 824, 32

Determination of Antibody Mechanism of Action Using IdeS Protease

Posted on by Gary Kobs

Monoclonal antibodies (mAbs) have been widely used to eliminate undesired cells via various mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and programmed cell death (PCD). Unlike the Fc-dependent mechanism of ADCC and CDC, certain antibody–antigen interactions can evoke direct PCD via apoptosis or oncosis. Previously, researchers have reported the specific killing of undifferentiated human embryonic stem cells (hESC) by mAb84 (IgM) via oncosis (1)

In a recent publication (2), a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC). One of the many experimental tools used to characterize the mechanism of oncosis was the fragmention of the A1 antibody with IdeS and papain.

Learn more about IdeS and IdeZ Protease available from Promega.

Papain digestion of IgG produces Fab fragments in the presence of reducing agent. F(ab)2 fragments of A1 were produced using IdeS Protease.

The results indicate that both Fab_A1 and F(ab)2_A1 bind to hESC but only F(ab)2_A1 retained hESC killing. Hence bivalency, but not Fc-domain, is essential for A1 killing on hESC.

  1. Choo, A.B. et al. (2008) Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1. Stem Cells  26, 1454.
  2. Zheng, J.Y. et al. (2017) Excess reactive oxygen species production mediates monoclonal antibody-induced human embryonic stem cell death via oncosis. Cell Death and Differentiation 24, 546–58.

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Hot Wings and Snow Birds: A Study of Genetic Selection in Chickens

Posted on by Sara Klink
African chicken breed Boschvelder. Image copyright ICBH GROUP.

This past summer, I visited the county fair and stopped by the animal barn to look at some of the poultry on display. Specifically, I wanted to see examples of the breeds of chickens available that I may be interested in adding to my flock. Rather than each chicken in their display cage being labeled with a bird’s breed, each cage listed the geographic origin of the chicken within such as Asiatic, Continental or American. This did not benefit my search for potential new members of my flock, but intrigued me enough that I wanted to find out how my flock of 19 hens and pullets would be characterized. Using the classes delineated by the Wisconsin State Fair, my feathered ladies break down to 12 American, 4 English and 3 Continental chickens. There are also classes for Mediterranean and Asiatic (and Other). I live in a part of the United States that gets cold, snowy weather for what seems like six months out of the year, weather that my chickens seem to take in stride. But in other places in the world, heat is the name of the game for the poultry strutting there. In a Genes, Genomics, Genetics publication, Fleming et al. wanted to know if there were genetic differences in Northern European and African chickens that might be caused by their environment.

Continue reading “Hot Wings and Snow Birds: A Study of Genetic Selection in Chickens”