Tuesday, June 20, 2017

Zebrafish Development: From a Few Cells to an Embryo

Zebrafish are a great model organism used to study numerous biological processes.  Some of the benefits of the zebrafish model are that they mature within a short period of time, they are optically transparent, develop outside of the mother’s body, and are vertebrates.  Because of these, and other reasons, zebrafish are used in a wide variety of applications including drug discovery, developmental biology, and molecular genetics. Zebrafish are easy to analyze in early development, as the embryo is relatively easy to manipulate and visualize.


Zebrafish embryonic development: from 16 cells to 1 day

The video above is a great example of this.  In the video, the development of a zebrafish embryo is highlighted, progressing from a few cells to a 1 day old embryo where the heart is beginning to beat and the blood is beginning to flow. This video was provided from a Cytation 5, with images taken every 2 minutes for 24 hours. The very first panel of the video shows an embryo with about 16 cells sitting on top of the yolk sac.  The embryo is surrounded by a protective membrane called a chorion.  For the first week of its life, the embryo will get all the nutrients it needs from the yolk sac. After the initial division, the cells of an embryo divide every 15 minutes.  Therefore, the cell number quickly progresses from 16 to 128 in one hour.  The cell mass forms a ball of cells on the yolk sac and this point marks the beginning of the blastula period.  During this part of development, the cells change from a high piled cell mound to a cup-shaped multilayer. This multilayer of cells begins to thin and spread over the yolk sac, and the embryo changes shape from spherical to oblong.

16 cell zebrafish embryo
16 cell zebrafish embryo
The next stage of development is gastrulation. Gastrulation starts at 50% epiboly, or when the cell layer is covering about half of the yolk sac. This is the stage when the primary germ layers (endoderm, mesoderm, and ectoderm) are formed. The gastrula period is over when epiboly is complete (i.e. when the cell layer is covering the whole yolk sac) and the tail bud has formed marking the posterior of the embryo. 
Zebrafish embryo during segmentation period
Zebrafish embryo during segmentation period
The next portion of development is my favorite; it is known as the segmentation period. This is when the embryo starts to look like something more than a bundle of cells and the eye and somites begin to appear.  Somites are blocks of mesoderm that form along the anterior-posterior axis of the embryo that will eventually give rise to skeletal muscle, vertebrae, and skin.  Halfway through the video you can see the eye in the upper right hand corner of the embryo, the tail in the bottom right hand side of the embryos, and the somites forming along the body. After the somites appear, you will notice that the embryo body begins to lengthen and eventually the embryo begins to move for the first time.  By the end of the video pigmentation has started to develop on the eye and in the skin and the embryo is at the beginning of the pharyngula period. The heart is just beginning to beat, the blood is beginning to circulate and the embryo is quite active. In just 24 hours the zebrafish embryo goes from a few cells to a live, moving, organism!

For more information check out:

By: BioTek Instruments, Sarah Beckman, PhD., Microscopist

Thursday, March 30, 2017

BioTek at AACR 2017

The American Association for Cancer Research (AACR) will hold its annual meeting next week from April 1-5 in Washington D.C. The meeting will celebrate an important anniversary and marks a homecoming of sorts as the association was founded 110 years ago in Washington D.C. This conference represents the largest gathering of cancer researchers and clinicians from around the world, as each year more than 18,000 people attend to learn about new advances within oncology.

One area promising to receive a great deal of interest this year is the field of immuno-oncology. Here the goal is to stimulate a patient’s own immune system to fight the specific cancer. Therefore, new drugs, or immunotherapies, are developed to target not the individual cancer cells, but particular cells of the immune system, such as natural killer (NK) or cytotoxic T lymphocytes (CTLs). BioTek will present an Exhibitor Spotlight Tutorial in cooperation with Lonza and Greiner Bio-One entitled “Demonstration of High Throughput, Image-Based 2D and 3D Natural Killer Cell Mediated Cytotoxicity Assays” to illustrate how in vitro immunotherapy optimization experiments can be performed in a walk-away manner and analyzed using highly sensitive cellular imaging. The tutorial will take place on Tuesday, April 4th from 4-5:00pm in Spotlight Theater B. We invite you to attend the talk and to stop by our booth (1857) to learn about all the 2D and 3D oncology applications optimized using BioTek’s instrumentation.

3D HCT116 tumoroid exhibiting apoptosis and necrosis following NK cell exposure.
3D HCT116 tumoroid exhibiting apoptosis and necrosis following NK cell exposure.
By: BioTek Instruments, Brad Larson, Principal Scientist

Thursday, March 2, 2017

Free up some time: 2017 SLAS

This was my first year attending the SLAS meeting. I was impressed with the tremendous breadth of automated instrumentation available to life science professionals. Being a bit of a gear-head, it was easy being captivated by all the moving arms, spinning pumps, traveling vessels and liquids being moved about. At the end of the day though, it really comes down to determining the bottlenecks in a particular workflow and how this myriad of technologies helps free up more time to manage other tasks.
BioTek Instrumentation at SLAS

Spending time listening to other scientists and automation partners visiting the BioTek booth provided excellent examples of how BioTek instruments are currently being used and ideas for future use as both standalone workstations or by incorporating into larger automated work cells. Examples ranged from an automated washing and dispensing workstation for up to 100 plates per day to live cell experiments lasting weeks by incorporating an EL406 Washer Dispenser and BioSpa Live Cell Imaging System to manage media exchanges and kinetic imaging/plate reading.

It appeared that once again the BioSpa was a big hit drawing the attention of many passers-by. Folks were pleased to find an automated solution with such a small footprint, intuitive control and scheduling software and flexibility to interface with a variety of instruments. Of equal interest was the Lionheart FX Automated Live Cell Imager for the same small footprint and integrated environmental control for longer term kinetic experiments on cells.

BioTek booth
As a testament to the practical use of the BioSpa System the Scientists’ Choice Award for Best Drug Discovery & Development Article included the use of the system in combination with an EL406 for research into cancer-causing genetic abnormalities and potential therapies.

Customers at BioTek booth at the SLAS
The meeting was a fantastic opportunity to connect with partners, customers and scientists working in a variety of areas, solving difficult scientific problems. Having a full complement of BioTek instruments available at both the BioTek and partner booths allowed for demonstration of possible solutions.

By: BioTek Instruments, Peter J. Brescia Jr., MSc, MBA

Thursday, February 16, 2017

Rigor and Reproducibility

Rigor and Reproducibility

A recent report found that the published findings from 89% of preclinical studies in the field of cancer research – in particular, those deemed ‘landmark’ studies – were unable to be confirmed by other scientists [1]. The authors concluded that despite competent, well-intentioned researchers, an alarming number of these inconsistencies were due to limitations in the design and execution of the initial studies, including the use of problematic endpoint measures and biased analysis of data.

This report is not unique to one particular research field, but is in fact representative of a growing awareness within the greater scientific community of the critical need for robust and unbiased experimental design [2-5]. The National Institutes of Health (NIH), one of the world’s leading medical research centers and major provider of extramural biomedical research funds, recently implemented new grant application instructions intended to enhance reproducibility through rigor and transparency [6]. Research grant applicants must now address four areas of focus:  scientific premise, scientific rigor, biological variables, and authentication.

The NIH defines scientific rigor as “the strict application of the scientific method to ensure robust and unbiased experimental design, methodology, analysis, interpretation and reporting of results.” This standard has been particularly difficult to achieve for imaging-based studies, which conventionally depend heavily on manual methods for collecting and analyzing data. This time-consuming approach frequently offers limited quantitative results due to small sample size and qualitative endpoints. Additionally, subjective selection criteria and lack of standardization across methods and instrumentation impedes reproducibility and transparency.

Recent advances in automated imaging systems have provided a great leap forward in our ability to conduct robust imaging-based biomedical research. The efficient and consistent acquisition of high quality images by BioTek’s Lionheart FX and Cytation automated imagers, across large numbers of conditions and replicates, allows researchers to meet the high standards set for reproducibility and transparency. Furthermore, the routine application of powerful Gen5 processing and analysis tools across entire image sets enables robust and meaningful quantitative results. 

Access to innovative technology and the prospect of life-improving therapeutics on the horizon make this an exciting time to be a scientist. Successfully translating biomedical research into clinical advances requires rigorous and reproducible approaches.  In our rapidly evolving scientific landscape, the need to demonstrate the relevance and benefit of sound scientific research is as important now as ever before.

  1. Begley, C.G. and L.M. Ellis, Drug development: Raise standards for preclinical cancer research. Nature, 2012. 483(7391): p. 531-3.
  2. Begley, C.G., A.M. Buchan, and U. Dirnagl, Robust research: Institutions must do their part for reproducibility. Nature, 2015. 525(7567): p. 25-7.
  3. Begley, C.G. and J.P. Ioannidis, Reproducibility in science: improving the standard for basic and preclinical research. Circ Res, 2015. 116(1): p. 116-26.
  4. Kretser, A., D. Murphy, and J. Dwyer, Scientific integrity resource guide: Efforts by federal agencies, foundations, nonprofit organizations, professional societies, and academia in the United States. Crit Rev Food Sci Nutr, 2017. 57(1): p. 163-180.
  5. Pusztai, L., C. Hatzis, and F. Andre, Reproducibility of research and preclinical validation: problems and solutions. Nat Rev Clin Oncol, 2013. 10(12): p. 720-4.
  6. Health, N.I.o. https://grants.nih.gov/reproducibility/index.htm. 2017.

By, BioTek Instruments, Joe Clayton, PhD., Principal Scientist

Tuesday, November 1, 2016

Anxiety and the U.S. Election

U.S. election season is reaching its zenith as we find ourselves in an endless tug of war between local and national political candidates, each vying for our vote. Emotions, including anxiety, are running high among candidates, electorates, and even from other countries that are not involved in the election process. As scientists, we appreciate the calming effects of facts and data, so we decided to detach from the day-to-day election buzz to take a look at how our instruments are being used to characterize anxiety in the laboratory.

Modulation of Anxiety and Fear via Distinct Intrahippocampal Circuits
In characterizing mouse hippocampal sub-regions, these researchers demonstrate separate regions of interest that are specific to suppressing anxiety and fear. Total RNA from these regions was measured using the Synergy HT Multi-Mode Reader.

Acute Stress Dysregulates the LPP ERP Response to Emotional Pictures and Impairs Sustained Attention: Time-Sensitive Effects (direct pdf download)
When ascertaining the effects of acute stress on subsequent emotion processing following an acute stressor, the ELx800 Absorbance Reader was used to measure salivary cortisol levels in female undergraduate students.

Anxiety, Depression, Stress, and Cortisol Levels in Mothers of Children Undergoing Maintenance Therapy for Childhood Acute Lymphoblastic Leukemia
Salivary cortisol levels were also measured, this time using a PowerWave 340, in mothers of healthy children, and also mothers of children with leukemia as part of a study to compare anxiety, depression and stress among these groups.

Regulator of Calcineurin 1 Modulates Expression of Innate Anxiety and Anxiogenic Responses to Selective Serotonin Reuptake Inhibitor Treatment
Using mice, regulator of calcineurin 1 (RCAN1) is positively identified as influencing expression of anxiety-related and selective serotonin reuptake inhibitor-related behaviors, which makes it a possible therapeutic target for anxiety. Calcineurin phosphatase activity in mouse prefrontal cortexes was measured using a Synergy 2.

We certainly feel less anxious now, but if those weren’t enough to calm you this election cycle, you can try a distraction technique. Here’s a picture of a basket of kittens. Enjoy!


By, BioTek Instruments

Monday, October 10, 2016

CRISPR: From editing to imaging

Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats) pathway, the evolution of the gene editing technology has been astounding. Applications now include not only an easily implemented method for genetic modification but adaptation for gene knockdown, generation of allelic series for control of differential expression, an alternative method for generation of inducted pluripotent stem cells, up-regulation for genetic screening and more recently loci imaging by creation of fluorescent protein:dCas9 conjugates to investigate nuclear and chromatin structure and dynamics with real time imaging1. While not without limitations the current applications show how quickly a technology can be adopted and further adapted to meet the needs of a plethora of investigators. In a similar fashion, the range of applications performed on BioTek’s expanding range of Automated Cell Imagers and Cell Imaging Multi-Mode Readers has been broadened. In a few short years the applications group and customer base have performed a wide range of assays such as wound-healing, proliferation, 3D cell culture and label-free cell counting, just to name a few. As the application space evolves so does the need for instrumentation that can provide flexible and affordable solutions. BioTek’s range of imaging capable instruments has certainly filled the bill. Wouldn’t automated loci imaging be interesting...


For further details of the imaging application space visit the BioTek website: http://www.biotek.com/resources/

For a video of live cell imaging by Drost, J. et. al.: http://www.nature.com/nature/journal/v521/n7550/fig_tab/nature14415_SV4.html

1. Drost, Jarno; van Jaarsveld, Richard H.; Ponsioen, Bas; Zimberlin, Cheryl; van Boxtel, Ruben; Buijs, Arjan et al. (2015): Sequential cancer mutations in cultured human intestinal stem cells. In Nature 521 (7550), pp. 43–47.

By: BioTek Instruments, Peter J. Brescia Jr., MSc, MBA

Monday, September 19, 2016

The Evolution and Domestication of Yeast

Anyone who knows me is aware that I’m a beer guy. While I’m a cell biologist by training one of my passions for quite some time is beer; whether it be tasting or brewing the beverage. In a recent article in the journal Cell, a group led by geneticist Kevin Verstrepen at the University of Leuven sequenced the genomes from 157 strains of Saccharomyces cerevisiea used to make ale, wine, sake, and bread [1]. When beer and science come together only good things can happen.

Yeast has been used to make fermented beverages for thousands of years. A 5,000-year-old Sumerian tablet describes an ancient party where ingredients to make fermented beverages known as beer today were used. Probably the first record of a kegger party! Since that time strains of S. cerevisiea have been developed to meet different needs.

Domestication of industrial yeasts
Figure 1.  Representation of the history and domestication of yeast used form making beer and other types of alcohol as revealed through genotypic and phenotypic analysis. Credit: Gallone and Steensels et al Cell 2016

The researchers dated the earliest cultivated yeast strains to the 1500s, which is likely a consequence of beer production in Europe moving from pubs into monasteries (Fig 1). As these early brewers fine-tuned their recipes, they also selected for favorable yeast strains. Domesticated yeasts have a greater capacity to metabolize sugar, fewer distasteful byproducts, and weaker reproductive abilities, compared to their wild-type cousins.

What is interesting is that the industrial yeast used today came from only a few ancestral strains. Five large groups separated out genetically, with strains mainly clustered together according to their industrial purpose. Geographic boundaries further divided each category: in one grouping of beer yeast, for example, the strains from Belgium and Germany were closely related, but separate from those in the UK and US [2].

As a brewer I know that the flavor of the beer depends greatly on the yeast. While many different beers can be made with the same strain using different grain mixtures, some beers that have very specific traits, such as the smoky clove and banana flavoring of German Hefeweizen, require specific strains. Hefeweizen requires the production of the compound 4-vinyl guaiacol (4-VG) in order to impart these unique flavors. These same flavors are considered flaws in other beer types. The genomes of the strains used to make Hefeweizen contain stretches of DNA, including the genes that make 4-VG, that seem to originate from wine yeast. It has been speculated that these strains emerged when an ale strain hybridized with a wine-making yeast, regaining the capacity to make the clove-smelling chemical.

Another point is that wine yeasts, which share their origins with beer yeast, show fewer signs of domestication. "This is probably because wine yeasts are only used to ferment grape juice once a year, and survive in and around the winery for the rest of the year, where they may interbreed with feral yeasts," Brigida Gallone and Jan Steensels of the University of Leuven told Scientific American.  "In that sense, beer yeasts are like dogs, completely 'tamed' and adapted to their relation with humans, whereas wine yeasts resemble the wilder character of cats." [3].

Me, I’m a beer guy who likes cats...

  1. B. Gallone and J. Steensels et.al (2016) Domestication and Divergence of Saccharomyces cerevisiea Beer Yeasts, Cell, 166(6):1397-1410.
  2. http://phys.org/news/2016-09-beer-yeasts-dogs-wine-cats.html#jCp
  3. http://www.scientificamerican.com/article/ale-genomics-how-humans-tamed-beer-yeast/

By: BioTek Instruments, Paul Held, PhD., Laboratory Manager