Author: Johanna Lee

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

Evaluating DNA Quantity and Quality in FFPE Tumor Samples After Prolonged Storage Using the ProNex® DNA QC Assay

Posted on by Johanna Lee

When tumors are surgically removed from cancer patients, the tumor samples are often stored as formalin-fixed and paraffin-embedded (FFPE) tissue blocks. In many cases, tumor samples need to be analyzed several years after diagnosis in order to develop target treatments. But what happens to the DNA after years of storage in FFPE blocks? How well is the DNA preserved?

Scientists in France tried to answer this question in a recent study published in Virchows Arch. The authors extracted DNA from 46 FFPE tumor samples of lung, colon and the urothelial tract, all stored between 4–6 years at room temperature. They then compared the quantity and quality of the DNA to DNA that had been extracted before storage. Using common fluorimetry and qPCR methods, the authors found that the total amount of DNA extracted decreased by half. In addition, the percentage of amplifiable DNA decreased from 56% to only 15% after prolonged storage. Continue reading “Evaluating DNA Quantity and Quality in FFPE Tumor Samples After Prolonged Storage Using the ProNex® DNA QC Assay”

Your Kid Can Become a Citizen Scientist with These 6 Apps

Posted on by Johanna Lee

Has your kid ever asked you what you do in the lab all day? (“Hmm…good question, what am I doing all day?”) A simplified answer might sound something like this: I observe, ask a question, collect data, and use those data to answer the question (or at least try!). The scientific method may be difficult to explain to a kindergartner, but you can always start by encouraging them to observe the world around them, ask lots of questions—and even help collect data. In fact, with the help of technology, your child can become a scientist without 7 long years in graduate school or ever setting foot in a lab. A “citizen scientist”, that is. All you need is a smart phone. Here is a list of apps that can make your kid, grandma, neighbor, anyone, become a citizen scientist by helping professional scientists collect data for their research. The apps are all free to download, easy to use and have a real impact on the scientific community.

1. iNaturalist:

Are you sick of not knowing the answer when your child asks “What’s this bug?” on a nature walk? You need the iNaturalist app. Here’s how it works: You observe an interesting plant/insect/animal, take a photo, and the app identifies the name of the species and some basic information about it. There is also the option to share your finding with other users and they can suggest an identification. iNaturalist shares your findings with scientific data repositories like the Global Biodiversity Information Facility to help scientists understand biodiversity. You can even set up scavenger hunt-like activities: iNaturalist birthday party anyone?

Continue reading “Your Kid Can Become a Citizen Scientist with These 6 Apps”

Measuring Metabolic Changes in T cells with the Lactate-Glo Assay

Posted on by Johanna Lee

Immunometabolism

Welcome to the emerging frontier of immunometabolism. A decade ago, immunology and metabolism were seen as two distinct areas of study. However, we now know that specific metabolic activities are required for proper immune cell differentiation and function. In tumor microenvironments, immune cells may even alter their metabolism to compete with tumor cells for limiting nutrients.

Glucose metabolism in Naïve vs Effector T cells

What does your car and T cells have in common? They both shift gears! You can shift gears on your car to change the way the engine’s power is used to match driving conditions; when you’re going uphill, you switch to a higher gear. Similarly, when T cells are activated, they change the way they generate energy to match functional needs. This makes sense because activated T cells (known as effector T cells) require more energy and biomass to support growth, proliferation and effector functions.

While cars run on gas, the main fuel for T cells is glucose. Each glucose molecule is broken down into pyruvate while generating 2 ATP molecules. Naïve T cells completely oxidize pyruvate through oxidative phosphorylation to generate 36 ATPs per glucose molecule. However, when T cells are activated and become effector T cells, glycolysis is used to produce 2 ATPs per glucose molecule. Continue reading “Measuring Metabolic Changes in T cells with the Lactate-Glo Assay”

iGEM: Saving the World with Science

Posted on by Johanna Lee

The University of Chicago 2016 iGEM team group photo (Photo credit: Julia Byeon)

Every year, groups of teenagers gather together and brainstorm ways to save the world—with science. The International Genetically Engineered Machine (iGEM) Foundation is a non-profit organization that is dedicated to educating young scientists and enhancing open community and collaboration in the field of synthetic biology. They hold a competition every year with hundreds of teams participating from around the world.

Last year, Promega provided cloning reagents to the University of Chicago iGEM team, and they received a bronze medal for their work. We asked two of the team members, Steve Dvorkin and Julia Byeon, about their experience. Steve is a junior and majors in biology; he is co-president of the team this year. Julia recently graduated and works in public policy. Continue reading “iGEM: Saving the World with Science”