Take Notes and Graduate Faster!

Cell density illustrationOne piece of advice you will get from our Technical Services and R&D Scientists with regard to cell-based assays is to pay attention to what you are doing. Sounds obvious, but sloppiness can easily enter into the equation. Do you always count your viable cells with a hemocytometer and trypan blue exclusion before you split a culture? Do you always make sure that each well of your plate or plates contain the same number of cells? Two of our scientists, Terry Riss and Rich Moravec, published a paper demonstrating how decisions you make in experimental setup can ultimately affect the results you obtain. A natural consequence of this is difficulty replicating experiments if you didn’t pay attention to the details during the initial experimental setup.

Cell Density Per Well Affects Response to Treatment
To demonstrate how cell density can affect your data, Riss and Moravec set up parallel plates with three different cell densities of HepG2 cells and measured the response to tamoxifen. The lower the cell density per well, the more pronounced the effect of the tamoxifin on the cells. Higher density cells were more resistant to tamoxifen. Continue reading

Choosing Primary and Control Reporters for Dual-Luciferase Assays

Dual-Reporter Assays give scientists the ability to simultaneously measure two reporter enzymes within a single sample. In dual assays, the activity of an experimental reporter is correlated with the effect of specific experimental conditions, while the activity of a control reporter relays the baseline response, providing an essential internal control that reduces variability caused by differences in cell viability or transfection efficiency. The Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay provides a choice of two sensitive reporters (firefly and NanoLuc luciferases) for use in dual-assay format. Both reporters give state-of-the-art functionality, raising the question “Which luciferase should be the primary reporter and which should be the control?”

This infographic outlines the various NanoDLR dual-reporter assay choices and the situations where you would choose one format over another. Continue reading

Thawing Out to Sing: The Story of the Wood Frog

Wood Frog_Northern WisconsinOne of the hallmarks of the arrival of Spring in Wisconsin is the cacophony of evening croaks and calls from the Spring Peepers and Chorus frogs. Indeed frogs and toads are ubiquitous around the globe, and many of us who have become life scientists (even those of us who have relegated ourselves to the world of macromolecules, cell signaling networks, and nucleic acids) probably spent some time in our childhood chasing and catching frogs.

But what happens to those frogs and toads over the harsh winter months in places like Wisconsin? Well, their strategies are species-dependent, but at least some of them overwinter by freezing, and the story of one species, the Wood Frog, is quite amazing. Think about it. It freezes from the inside out. No heart beat, no circulation, completely dormant. Then in response to some unknown signal (day length? temperature? angle of the sun?), bodily functions slowly resume. What kind of cell signaling cascade controls that response?

Here are a couple of videos about the Wood Frog and its amazing deicing event. The next time you are out on a Spring or Summer evening and you hear a chorus of frogs calling, you can think about the incredible molecular story behind the event and be even more impressed!

A NOVA Video about the Wood Frog:

A Science 360 video on the Wood Frog of Alaska

Do You #Authenticate?

authenticate_logoOn April 15, 2015 Nature announced a new policy around authentication of cell lines used in research studies that are published in its journals (1). Beginning in May 2015 they are asking all authors to confirm that they are not working on cells known to have been misidentified or cross-contaminated and to provide details about the source and testing of their cell lines.

The problem of cell line misidentification has been well documented in the literature with issues being reported with hematopoietic cell lines in 1999 (2) and a lymphoma cell line in 2001 (3). In 2006, one study suggested that 15–20% cells used in experiments have been misidentified or cross contaminated (4). And, in her book The Immortal Life of Henrietta Lacks, published in 2010, science writer Rebecca Skloot, notes that concern about cell line contamination dates back to 1958 (5). Promega has written about this problem and the power of STR analysis to assist you in assuring that your cell lines are what they should be (6–9). In fact, as John Masters, Professor of experimental pathology at University College, London points, out, there really is no excuse for the continuing problem of cell line contamination:

“For nearly 50 years, people have been using falsely identified cells totally unnecessarily because they haven’t checked.” (10)

Know what your cells are. #authenticate

Know what your cells are. #authenticate

The problem of cell line misidentification and contamination is not a new problem, and the calls for the scientific community to take extra care in understanding the identity of the cells that they are working with are not new either. Nature journals are not the first journals to take a stand to require authors to authenticate their cell lines. Journals including  International Journal of Cancer , In Vitro Cellular and Developmental Biology and Cell Biochemistry and Biophysics previously put policies in place around this issue (11,12), and in 2012 a new standard (ASN-0002) was officially published by the American National Standards Institute regarding human cell line authentication using profiles generated from STRs (11) . Based on the work of the ASN-0002 work group, the International Cell Line Authentication Committee was formed to promote awareness and authentication testing worldwide (13), including creating a publicly available database of misidentified cell lines.  However, as more and more high-profile cancer studies are retracted because of cell line issues (14,15), it has become apparent standards for cell line culture and authentication will need to become common place in life science research. Continue reading

Animal or Plant? FISH Labeling Reveals Horizontal Transfer of Algae Gene into Sea Slug Chromosome

There are times when I ask myself why I chose a career in science. This happens on what I call “grass is greener” days. On these days I dream of other careers—like National Geographic reporter or Caribbean tour guide–which all sound way more exciting than scientist. Admittedly these alternative careers are not ones that many people have the privilege of attaining, but sometimes reality gets to take a vacation. Fortunately, science is a fast-moving, always-changing field. As much as I might occasionally dream of exotic jobs in far away locations, science always pulls me back in with something new and unexpected. Because as much as we’d like to think we know, the truth is there is so much more that we don’t.

Image from: Pelletreau KN, Weber APM, Weber KL, Rumpho ME (2014) Lipid Accumulation during the Establishment of Kleptoplasty in Elysia chlorotica. PLoS ONE 9(5): e97477. doi:10.1371/journal.pone.0097477

The sea slug Elysia chlorotica. Image from: Pelletreau K.N., et al. (2014)  PLoS ONE 9: e97477.

A case in point—sea slugs. These unfortunately named, exotic looking creatures have some surprising secrets. Continue reading

Rapid Evolution in Response to Rapid Climate Change?

21491767_lIn celebration of Earth month, Environmental Education Week, Earth Day and National DNA Day (celebrated this week in many nations),  we have a Science 360 video that highlights the work of Drs. Bradshaw and Holzapfel to explore climate change-driven genetic shifts in the Pitcher Plant Mosquito. It’s a fascinating and thought-provoking question these scientists are trying to answer. You can read more of the latest work at their laboratory web site.  What are you doing to celebrate Earth Day/Environmental Education Week activities?

Removing Cancer’s Cloak of Invisibility

Copyright Promega.

Copyright Promega.

For decades scientists have been trying to harness the power of our immune system to fight cancer cells. It is not impossible to imagine that our immune system, which is sophisticated enough to fight against a multitude of invaders that threaten our health, should be able to tackle a deadly disease such as cancer. This formed the basis of testing a new type of cancer treatment known as immunotherapy. Immunotherapy for cancer means developing treatments to harness your immune system and using your own immune system to fight the cancerous cells.

But in reality it was hard to make this work. Because, as scientists discovered recently, cancer outsmarts the immune system by wearing a kind of “invisibility cloak”. Cancer is able to fool the immune system from recognizing that it is the enemy and in effect keeps the immune system from destroying it.

In a breakthrough discovery scientists have found a way around this treachery.

The breakthrough is in therapies called ‘checkpoint inhibitors’. Checkpoint inhibitors block the mechanisms that allow some tumor cells to evade the immune system. The drugs ensure that cancer cells are no longer be shielded from the immune system defenses, but are instead recognized as “foreign”. Continue reading

Cloning Tips for Restriction Enzyme-Digested Vectors and Inserts

Cartoon created and owned by Ed Himelblau

While T-vector cloning is commonly used for PCR-amplified inserts, restriction enzymes still have their uses. For example, you can ensure directional cloning if you digest a vector with the same two enzymes like BamHI and EcoRI that are used to digest your insert. This way, the insert can only be cloned in one direction. However, there are other cloning techniques that can be used to modify the end of vectors and inserts after restriction enzyme digestion and prior to ligation. Continue reading

HaloTag® Research Application: Detection of Cancer Biomarkers

10242TAAntibodies labelled with radioisotopes or the sequential administrationof an antibody and a radioactive secondary agent facilitate the in vivo detection and/or characterisation of cancers by positron emission tomography (PET) or by single-photon emission computed tomography (SPECT) imaging.

There are drawbacks to both methods, including prolonged exposure to radiation and  ensuring that both the antibody and the radiolabelled secondary agent are suitably designed so that they bind rapidly upon contact at the tumor.

A recent publication (1) investigated a alternative method utilizing the HaloTag® dehalogenase enzyme HaloTag® is a dehalogenase enzyme (33 kDa) that contains an engineered cavity designed to accommodate the reactive chloroalkane group of a HaloTag® ligand (HTL). Upon entering the enzyme cavity, the terminal chlorine atom rapidly undergoes nucleophilic displacement, and a covalent adduct is formed, effectively anchoring the HaloTag® ligand in a precise location.

Three new HaloTag® ligands were synthesized and each labelled with the SPECT radionuclide indium-111  111In-HTL-1  and the dual-modality HaloTag® ligands,111In-HTL-2 and111;In-HTL-3 containing TMR which allows complementary imaging data).

For the validation of the pretargeting strategy based on these HaloTag® ligands, the target human epidermal growth factor receptor 2 (HER2)was selected. Trastuzumab (Herceptin®) was selected as the primary targeting agent and was modified with HaloTag® protein via the trans-cyclooctene/tetrazine ligation.

All three 111In-labelled HaloTa®g ligands exhibited significantly higher binding to the HER2 expressing when compared to negative controls.

Literature Cited

Knight, J. C et al.(2015) Development of an enzymatic pretargeting strategy for dual-modality imagingChem. Commun. 51, 4055–8.

A New Way to Avoid False Hits During Compound Screening for Drug Discovery

One goal of drug discovery and research programs is to reduce false hits as early as possible in the process. Follow-up on false hits is costly in terms of time and resources, and the longer the false hits remain in the drug development pipeline, the more costly they are. So methods that can easily reduce the number of false hits during compound screening early in the discovery process are particularly sought after.

Reporter assays have proven to be invaluable tools for elucidating the mechanisms of action of small molecules or other agents on signaling pathways within cells, and the luciferase reporter assay has become a standard research tool in the biological research laboratory.

However, one caveat of using standard luciferase-based reporter assays for larger-scale compound screening efforts is the frequency of false hits that result from direct interaction of compounds with the luciferase reporter. This issue can be mitigated with a “coincidence reporter” system where two independent reporter proteins are produced from a single transcript. In this type of assay, a bicistronic transcript is stoichiometrically translated into two nonhomologous reporters by means of a 2A “ribosomal skipping” sequence. Since it is unlikely that compounds will interact with two distinct types of reporter, “coincident” responses will indicate on-target activity. Such a coincident reporter system provides an important control against costly false hits early in drug discovery research programs.

A paper published online in ACS Chem Biol in February describes the first successful application of the firefly/NanoLuc luciferase coincidence reporter system to identify new pathways that up-regulate PARK2 expression. Continue reading