Tuesday, June 21, 2016

Using Automated Imaging and Cellular Analysis to Increase Throughput and Objective Result Generation in Genotoxicity Assays

Genotoxicity refers to the potential of in vivo or ex vivo agents, such as cancer-causing cellular mechanisms, environmental compounds, or chemical molecules to induce damage or alterations to DNA, genes and chromosomes. Testing for potential inducers that cause, or treatments that can repair DNA damage has implications for a number of life science research areas, including oncology, environmental, and cosmetic research. Several in vitro genotoxicity tests have been developed, and among the more popularly performed tests are the Ames test which uses histidine negative Salmonella bacterial strains; the micronucleus test where treated or test cells are visually examined for the presence and frequency of micronuclei created due to toxicity, the single cell gel electrophoresis (SCGE) or comet assay where fragmented (damaged) and undamaged DNA create a comet-like configuration in a gel upon electrophoresis and staining; and the gamma H2AX (γ-H2AX) assay where nuclear foci created from antibody labeled phosphorylated histone 2AX are a biomarker for genotoxic exposure.

The desire to perform genotoxicity tests in a higher throughput format has increased as a direct relationship to the incorporation of some of these tests to develop personalized medicines or to move away from the use of animal models. This is witnessed by the growing number of companies offering standardized "off-the-shelf" plates, kits, and reagents to perform these assays. In addition, contract research organizations (CROs) are now offering their services in this area to increase throughput.

While methods to improve throughput have advanced recently, the means to perform these higher throughput assays has in many ways stagnated. Imaging is still performed manually in many situations. Analysis can also be a daunting task, particularly in the case of the comet assay where manual hunting for the “correct” comet is performed, images are taken, and the process is then repeated…comet after comet, and well after well. Not only is this process extremely time consuming, but also builds in a great deal of subjectivity into the analysis. BioTek's Cytation™ 5 and Lionheart™ FX automated imaging systems and Gen5™ Microplate Reader and Imager Software can provide easy-to-use solutions to both automate the image capture process and create objective analyses of genotoxicity assays.

The first example shown below illustrates imaging and analysis of the comet assay. Following fluorescent staining, the Cytation 5 Cell Imaging Multi-Mode Reader was used to automatically image the samples. Unlike manual methods, simple and advanced inclusion/exclusion criteria were programmed into the Gen5 Microplate Reader and Imager Software to automatically place object masks around each separate comet head and tail to eliminate anomalies and other false objects that might be mistakenly included in manual analyses (Figure 1). The total fluorescence intensity in the tail portion of the comet, in relation to the total comet fluorescence, is used to calculate “Percent DNA in the Tail”. These calculations can then be combined with object size data to calculate the “Comet Tail Moment”.
Automated comet analyses to determine (A.) comet head and (B.) comet tail in relation to the comet head.
Figure 1. Automated comet analyses to determine (A.) comet head and (B.) comet tail in relation to the comet head.
The second example illustrates a nuclear stain imaging and analysis of the γ-H2AX assay. Immunofluorescence using a primary antibody specific to γ-H2AX and a Cy-5 labeled secondary antibody is initially performed followed by automated imaging. Two separate cellular analyses are then carried out using Gen5 software. The first to determined the average area of a single labeled phosphorylation site, or foci. The second to place object masks around individual nuclei using primary analysis parameters, and then secondary parameters to place masks around individual foci within each nuclei (Figure 2). The combined analyses allow for a number of metrics to measure DNA damage to be generated, such as labeled foci coverage area and calculated foci number per nuclei.
Automated γ-H2AX analyses to determine (A.) cellular nuclei per image and (B.) labeled foci within each nuclei.
Figure 2. Automated γ-H2AX analyses to determine (A.) cellular nuclei per image and (B.) labeled foci within each nuclei.
By incorporating automated imaging and analysis many of the headaches and limitations of performing in vitro genotoxicity assays can be eliminated. Countless hours do not have to be spent in front of a manual imager and objectively generated results have greater comparability from experiment to experiment. We encourage you to contact BioTek to find out more about how these assays can be performed.

By: BioTek Instruments, Brad Larson, Principal Scientist

Friday, June 17, 2016

Multiple Sclerosis

Multiple Sclerosis (MS) is a disease that affects a number of my friends in various degrees.  It’s a demyelinating disease in which the insulating cover, known as the myelin sheath, of nerve cells in the brain and spinal cord are damaged.  The result as you can imagine can be very debilitating and can lead to death.  While it is not my field of study, I’m always on the lookout for articles regarding this disease.  Interestingly enough I came across two separate articles on the same day regarding the disease that on face value seem unrelated, but actually fit together.

The first article was in IFLScience by Graham Wright referencing a report in the The Conversation describing a mystery in MS rates of Multiple Sclerosis in Northern Scotland.  MS has been shown to be very prevalent on the Orkney isles, which lie off the northernmost mainland.  In these reports, studies supported the theory that low levels of vitamin D were a key initiating factor in the damage to neuronal transmission pathways.  The thinking is that because these islands are so far north access to sunlight is limited, resulting in less active vitamin D being produced.  The mystery is that while MS in Orkney is 402 cases per 100,000, compared to 200 in the rest of Scotland and 165 in England, the even more northerly Shetland isles have a rate of 295 per 100,000.  This indicates that more is going on than just sunlight exposure, with speculation as to a cultural shift between the two island groups and sun exposure.

Simplified Pedigree for Families Presenting the NR1H3 p.Arg415Gln Mutation

Figure 1. Simplified Pedigree for Families Presenting the NR1H3 p.Arg415Gln Mutation.  Two family trees that had the MS-causing mutation. “M” denotes individuals with the mutation, with black circles indicating individuals with MS.  Image courtesy of the University of British Columbia
The second was an article in Lab Manager reporting on a research paper in the journal Neuron describing the genetic cause of multiple sclerosis in two different families in Canada.  These families each had several diagnosed with the disease with a rapid progressive type.  The mutation found was actually a substitution of a single nucleic acid in the gene called NR1H3, which produces a protein known as LXRA.  This protein acts as an on/off switch for other genes that prevent excessive inflammation.

Like the Orkney isles, Canada has one of the highest rates of MS in the world, with an estimated 100,000 Canadians living with MS (286 cases per 100,000).  The obvious connection between the two groups is that the Scots are among the first Europeans to establish themselves in Canada and are the third largest ethnic group in the country. In the 2011 National Household Survey, a total of 4,714,970 Canadians, or 14.1 per cent of the population, listed themselves as being of Scottish origin. 

While these findings do not identify the root cause for all MS cases it does provide information on new research pathways, as well as provide information regarding new animal models.  Genetic screening might also provide information for earlier intervention (such as vitamin D supplementation) and more aggressive treatments for those identified as having the mutation.  I like to think that instrumentation provided by the company I work for, BioTek Instruments, can help with this research.

  1. Weiss, E. ,L. Zgaga,S. Read, S. Wild, M. G. Dunlop, H. Campbell, R. McQuillan, and J. F. Wilson  (2016) Farming, Foreign Holidays, and Viatmin D in Orkney, PLOS one, http://dx.doi.org/10.1371/journal.pone.0155633
  2. Zhe Wang, Z.,  A. D> Sadovnick, A. L. Traboulsee, J. P. Ross, C. Q. Bernales, M. Encarnacion, I. M. Yee,M. de Lemos, T. Greenwood, J. D. Lee, G. Wright, C. J. Ross, S. Zhang, W. Song, and  C. Vilariño-Güell (2016) Nuclear Receptor NR1H3 in Familial Multiple Sclerosis, Neuron, 90(5):948-954. DOI: http://dx.doi.org/10.1016/j.neuron.2016.04.039

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

Tuesday, May 24, 2016

How BioTek is Aiding the Cosmetics Industry in the Development and Testing of New In Vitro Non-Animal Based Epidermal Models

On May 11th I traveled to Finland to begin two weeks of scientific seminars at multiple customer sites across Europe. Many of the sites I visited in the south of France over the past week were dermatology companies interested in using BioTek's Cytation 5 Cell Imaging Multi-Mode Reader or Lionheart FX Automated Live Cell Imager to test new three dimensional (3D) epidermal models they are developing. This is in response to the complete ban on the sale of cosmetics developed through animal testing, which applies to all new cosmetics and their ingredients sold in the European Union, regardless of where the testing was carried out. To comply with these regulations, while still performing the testing required to deliver safe products to the public, dermatology and cosmetics companies are developing 2D and 3D skin, eye, mouth and other epidermal models using human derived cell lines, primary cells, and differentiated stem cells.

BioTek is aiding in this process by providing instrumentation and software solutions that allow streamlined, robust image-based tests to be performed with these new epidermal models.

Images captured by the Cytation 5
Detection of oxidative stress and hypoxia in immortalized keratinocyte cells using dual mask cellular analysis of images captured by the Cytation 5.
Analysis of wound healing using 3D bioprinted keratinocytes

Analysis of wound healing using 3D bioprinted keratinocytes and Gen5 cellular analysis capabilities. 
This combination results in cosmetic products that consumers can be confident will deliver the expected result and have been developed using methods free of animal testing.

By: BioTek Instruments, Brad Larson, Principal Scientist

Friday, March 18, 2016

Imaging and Analysis of Genotoxicity using Single Cell Gel/Comet Assay

On March 9, I had the privilege of co-presenting a webinar with Dr. Sachin Katyal from the University of Manitoba. The focus of the webinar was genotoxicity. Genotoxicity, or DNA damage, can be caused by environmental factors, behavior choices, or chemicals. Therefore it is important to test new chemicals in a wide range of applications (drugs, pesticides, dermatological agents, food additives, etc.) to ensure they do not cause damage to DNA. Or in the case of cancer look for treatments that can repair damaged DNA repair mechanisms. While there are many different types of assays to accomplish this goal, one of the most popular and most specific techniques is the comet assay. The term comes from the visual readout of the assay that resembles a telescopic image of a tradional astronomical comet that incorporates both the comet head and a long tail of streaming particles.  Conversely, the comet assay readout is produced after cell treatment with potential DNA damaging agents, mixed with agarose and added to an appropriate slide, lysed and alkaline treated, followed by electrophoresis. Upon staining with a DNA intercalating dye, cells with high levels of DNA damage will exhibit a similar comet shape (Figure 1, close up of a comet; and Figure 2, many comets in a single experiment to provide statistical significance).  These comet images are produced by the fact that fragmented DNA runs through a gel much easier than intact DNA strands. By incorporating the Cytation Cell Imaging Multi-mode readers and Gen5 software, a process that is typically very laborious and subjective can be automated to reduce manual interventions, and also have the subjectivity removed by performing analysis of all samples using mathematical algorithms. The final analysis method was extensively validated by comparisons to results generated using existing comet analysis software packages (Figure 3).

We were pleased to see 300 people register for the webinar, with many watching the actual event and participating in a lively question and answer session.

genotoxicity assays

We invite you to download the webinar recording and published application note from BioTek’s website to learn more about our combined solution to perform these critical genotoxicity assays.

By: BioTek Instruments, Brad Larson, Principal Scientist

Tuesday, March 8, 2016

Vaccines and my Daughters

Vaccines have revolutionized medicine since their inception by Edward Jenner in 1798. Diseases such as small pox or polio which used to kill millions have almost been eradicated using vaccines.   These two along with a litany of other diseases are prevented through a series of childhood vaccinations. With three kids, my wife and I (mostly my wife) made numerous trips to the pediatrician's office in order for one or more of my kids to get their "shots".

One of the more recent advancements in vaccine therapies has been the development of vaccines towards human papillomavirus (HPV).  HPV is one of the most common viruses on the planet -- most people don't even know they have it and it is often sexually transmitted. In some women, however, it can have concerning consequences as the virus causes abnormal cells in the cervix and can lead to cervical cancer if left untreated. Currently HPV is the etiologic agent in 5% of all cancers worldwide [1]. These vaccines, marketed under the names Gardasil and Cervarix, provide immunity from about 75% of HPV strains. Most importantly is that it protects against those strains that are known to cause cervical cancer. Unfortunately this vaccine does not help those already infected with HPV and that's the problem.

High Grade Squamous intraepithelial lesion Pap Test.
High Grade Squamous intraepithelial lesion Pap Test. Courtesy of Michael Bonert  [2].

The inventor of the vaccine, Ian Frazer, explains this "It's quite simple really, most viruses kill the cells they infect, which is a nasty danger signal for the body so it turns on its defenses pretty quickly to kill it and then kill the cells making more of the virus. This process saves us from flu and a whole range of different infections." Human papillomavirus doesn't kill the cells it infects -- rather it makes them grow more. There's no danger signal to body -- all the body sees is tissue repairing itself." [3].  Because of this, Professor Ian Frazer and others are working on a vaccine that uses viral proteins that are normally on the surface of the infected cell as immunogens rather than the coat proteins of the intact virus.  It turns out that a new type of vaccine seems to work best in animal models, namely DNA vaccines.

DNA vaccines are considered to be the third generation of vaccine technology, and contain DNA coding specific proteins (antigens) from a pathogen. The vaccine DNA is injected into the cells of the body, where the "inner machinery" of the host cells "reads" the DNA and uses it to synthesize the pathogen's proteins. Because these proteins are recognized as foreign, when they are processed by the host cells and displayed on their surface, the immune system is alerted, which then triggers a range of immune responses.

While both of my daughters received the HPV vaccine as children and are most likely protected, these new vaccine technologies have the potential to save millions of lives. This technology is being used primarily in animals and currently there are no DNA vaccines approved in the US, but the few experimental trials offer substantial promise. If this can be made to work it might be a viable therapy for a number of other diseases including some cancers.

  1. Parkin et. al. (2005) Global Cancer Statistics, 2002 CA Cancer J. Clin. 55:74-108.
  2. By Nephron - Own work, CC BY-SA 3.0,  (2010)  https://commons.wikimedia.org/w/index.php?curid=9084518
  3. Cayla Dengate, (2016)  “Gardasil Creator Is Testing A DNA Vaccine To Wipe Out Cervical Cancer-Causing HPV Virus” Huffington Post Australia, http://www.huffingtonpost.com.au/2016/03/01/cervical-cancer-dna-vaccine-hpv_n_9350280.html.

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

Tuesday, February 9, 2016

SLAS 2016 and BioSpa 8: A Love Story

Even though "SLAS 2016 and BioSpa 8" might not be a great title for the latest romance novel, it's a true blossoming love story we have witnessed at the SLAS meeting and exhibition that BioTek attends every year.

BioTek booth

With the explosion of live-cell biology applications, more and more long-suffering cell biologists are banging their heads on the wall with frustration. The pitfalls of working with live cells are many, and the task of performing live-cell experiments may appear daunting. Many have been seen on the verge of losing their mind after working for days or weeks on preparation work only to see it all go in a quick twist of fate: It takes amazingly little to contaminate a cell culture, or to drop samples on the floor after a bad night. The inevitable question is: There must be a better way, right? So much pain and suffering in the name of science begs for a solution. Surely there is a cure out there; someone has already figured it out. Unfortunately, the curious scientist trying to answer this question will quickly find himself navigating between Scylla and Charybdis, for the solution is often automation so monstrous it is best left in the hands of the valiant adventurer, helped with an army of highly trained lab technicians and automation professionals. What to do? On one hand Charybdis the whirlpool monster of manual cell work which will get your head spinning with its long nights and weekends, heartbreaking failures and gnawing uncertainty (what is happening to my cells when I am not around?); On the other hand, Scylla, the 6-headed monster of robotic automation, that will demand more attention than your cells themselves, and will require that you boost your software and hardware IQ by 50 points before you even dare to approach it.

Then one fresh spring morning (well, it was actually in the dead of winter, but this is a love story, right?), something happened: A solution seemingly came out of nowhere with the soothing name of “BioSpa 8”. Wait… this can’t be automation, it is way too small… but… this is clearly not manual, there is some kind of moving arm in front. This brings us back to reality, and what we heard from visitors stopping by our booth at SLAS 2016 to look at BioSpa 8 in action. It went like this:
  • "This is such a great idea!"
  • "I just saw your ad: it's even smaller than I thought."
  • "This price includes the arm too? Nice!"
  • "Does it record all parameters over time? Great! And it sends text notifications? Wow!"
  • "That's it? You just programmed it?"
With the BioSpa 8 Automated Incubator, there is a better way! You can still choose to navigate the perilous strait between Scylla and Charybdis if you enjoy the rush and the sleepless nights. Or you can try the BioSpa way. Let simple, small bench top automation soothe you. And you can go back home (at a decent hour) with the reassuring thought that your days of "are-my-cells-going-to-make-it?" anguish are finally over.

BioSpa 8 in BioTek's booth

By: BioTek Instruments, Xavier Amouretti, Manager, Product Marketing

Tuesday, January 12, 2016

Beyond the Solar System: Automated Comet Analysis

In our last comet assay blog post, ‘Beyond the Solar System: Automated Comet Assay Imaging’, we discussed how the comet assay  is used to directly quantify DNA damage in mammalian cells, and showed how to use Trevigen’s CometAssay® Electrophoresis System, 3-Well FLARE™ slides and 96-Well CometSlides with the Cytation 5™ Cell Imaging Multi-Mode Reader to easily image these comets. Here we’ll discuss how to analyze those images to quantify the extent of DNA damage. This quantification can be expressed as either the % DNA in the Tail or the Comet Tail Moment. Each method quantifies DNA damage by assessing the fluorescence from comet tails attributable to DNA fragments electrophoresing farther in the gel than intact DNA.

% DNA in Tail Calculation
This method is based on determining the relative circularity of the comet head and the total comet which may contain a comet tail if there is significant DNA damage. The following equation can be used:
DNA in tail
This analysis works well due to the fact that the total comet becomes less circular with increasing DNA damage. In the case of little DNA damage, it is apparent that Total Comet Circularity ≈ Comet Head Circularity, thus % DNA in Tail ≈ 0%.However in the case of extensive DNA damage, Comet Head Circularity >> Total Comet Circularity and % DNA in Tail will tend towards 100%.
We used Gen5 Data Analysis Software to apply this calculation to all images, and using the 96-well CometChip calculations (Table 1) as an example, we can see that % DNA in Tail values increased appropriately with increasing etoposide treatment (T1-T3) of the healthy Alkaline CometAssay Control Cells (T0). The % DNA in Tail values are comparable to those obtained via different methods like ImageJ open source software with the OpenComet plugin.
% DNA in tail calculations for 96-well CometChip
Table 1. % DNA in tail calculations for 96-well CometChip

comet head primary cellular analysis
Gen5 results compare well to those seen using Loats Analysis software using the 96-well CometSlide (Figure 1), further confirming that Cytation 5 and Gen5 deliver accurate results regardless of the configuration.
Percent DNA in tail calculations for 96 well CometSlide using Alkaline CometAssay Control Cells in the standard comet assay
Figure 1. Percent DNA in tail calculations for 96 well CometSlide using Alkaline CometAssay Control Cells in the standard comet assay

Comet Tail Moment
Another comet evaluation calculation is the Comet Tail Moment (Figure 2), which takes into account the Total Comet Length (DS1), Comet Head (DS2) and % DNA in Tail (DS3) as represented in the following equation:

Comet areas included in comet tail moment calculation
Figure 2. Comet areas included in comet tail moment calculation
Comets exhibiting little to no DNA damage will have a Comet Tail Moment value approaching zero, whereas those with higher damage amounts will have increasing values. Gen5 Data Analysis Software was again used for automatic calculations, and as seen in Table 2, the Comet Tail Moment values increased appropriately with increasing etoposide treatment (CC1-CC3) as compared to untreated cells. Furthermore, these results are equivalent to those obtained via accepted methods like Loats Analysis software.

Comet Tail Moment calculations for 3 well CometSlides.
Table 2. Comet Tail Moment calculations for 3 well CometSlides.

As Cytation 5 automates cellular analyses, DNA damage using the comet assay is rapid, repeatable and comparable to analyses obtained via different analyses methods. For more detail, see our application note, Automated Imaging and Analysis of a Novel Comet Assay to Enable High Throughput Genotoxicity Testing.
By, BioTek Instruments