Tuesday, November 24, 2015

Reactive Oxygen Species: a Mechanism of Action for Insulin Resistance and Type 2 Diabetes

About one third of the US adult population is defined as obese (BMI > 30 kg/m2). While not all obese adults develop type 2 diabetes, most (~80%) people suffering from type 2 diabetes are obese1. The links are clear: overeating and a sedentary lifestyle lead to obesity and in a significant number of these individuals, insulin resistance which is the hallmark for type 2 diabetes. The mechanisms producing insulin resistance have remained unclear, however. Researchers have proposed a number of possibilities, including elevated levels of fatty acids, inflammation, endoplasmic reticulum stress and oxidative stress through the production of reactive oxygen species (ROS).

In a recent study, six healthy middle-aged men were subjected to a whopping 6,000 calorie/day diet while being confined to a hospital bed2. Within 2 days of the start of the study, the men began showing signs of insulin resistance; after a week, the men demonstrated on average a 50% decrease in their insulin-stimulated glucose uptake - a clear sign of insulin resistance.  In that week, the men had gained an average of 3.5 kg - all of it fat. That fat was biopsied and tested for the possible mechanisms of insulin resistance outlined above. Only ROS production mirrored the dramatic increase of insulin resistance.   It was found that numerous proteins associated with ROS production were up-regulated along with oxidation and carbonylation of a wide range of proteins, notably of the protein GLUT4, which translocates from intracellular vesicles to the plasma cell membrane due to insulin signaling. This protein’s structure in the fat biopsies demonstrated multiple oxidation and carbonylation sites, particularly where the glucose binding site is thought to be. This would make the glucose transporter dysfunctional and thus lead to insulin resistance.

To learn more about ROS and its physiological consequences, please download our white paper An Introduction to Reactive Oxygen Species.

1. Eckel, RH et al. Diabetes Care June 2011 vol. 34 no. 6 1424-1430.
2. The 6,000-Calorie Diet

By: BioTek Instruments, Peter Banks Ph.D., Scientific Director

Wednesday, November 18, 2015

My Love Affair with the Spa; or How I learned to get my Life Back

For a number of years, I have been interested in the mammalian cell cycle. As a graduate student I worked on the relationship between the enzyme HMG CoA reductase and DNA synthesis.  HMG CoA reductase is the rate limiting step for cholesterol biosynthesis. It’s also the enzyme target for statins, which are one of the most prescribed classes of drugs in the US. Later as a post-doc my research focused on the initiation of DNA synthesis.  More recently I have been interested in the effect anti-cancer agents have on cell cycle progression.

What that really means is that I have suffered from sleep deprivation. If you have never worked on the mammalian cell cycle you might not understand the connection between cell cycle research and sleep deprivation. You see while some cells lines have longer cycles and others shorter, the average mammalian cell cycle time is approximately 24 hours long.  If you were interested in some sort of transient cell cycle regulator your experiments often required manual intervention (i.e. reagent addition, aliquot removal, measurement taken etc.) periodically throughout the entire cell cycle. The end result is that for the duration of the experiment, which might be two complete cell cycles, sleep was a commodity in short supply. On a Monday morning my first thought was that I could go to sleep on Thursday!

Sure there was automation available to those that could afford it. Large very expensive robotic systems were the rage for a period of time. Costing hundreds of thousands of dollars, these were just a dream to most researchers. Despite their price tag, most were not a viable option for live cell assays as they were not contained in HEPA filtered sterile environments.        

By now I’m sure you are wondering how this tale of woe is going to end; fortunately there is a happy ending. Much to my relief, BioTek developed the BioSpa 8. A fully functional, environmentally controlled robot that interfaces liquid handing devices with detection devices. Much like Cinderella, this solution fits me just like her glass slipper.  The BioSpa 8 holds up to 8 microplates (6- to 384-well), controls temperature, CO2, O2 and can be also provide a humidified environment in the same way as a conventional tissue culture incubator.  A robotic gripper arm can move plates from the hotel to either a BioTek liquid hander (e.g. MultiFlo FX dispenser, EL406 washer/dispenser, or a 405TS washer) or a BioTek reader (e.g. Cytation 3/5, Synergy Neo2 or an Epoch 2).  By interfacing these two device types with a plate hotel and scheduling software, a multitude of assays can be automated without breaking the bank.  The size of the system is such that it can fit in a 6 foot biosafety cabinet, allowing sterility to be maintained for live cell based assays.

Instead of spending my days and nights in the lab running experiments I can go home knowing that the reagent will get added, the samples will be washed and the plate will read.  Yes I got my life back…

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

Thursday, October 8, 2015

Analyzing the progression of the “Blood Moon” Eclipse with Gen5 software

When given the option of witnessing a once in a life time spectacle or going to bed like a responsible adult... well... usually my inner child kicks in to make the decision, which means my "responsible adult" self gets the night off.  And so it was with the recent Blood Moon Eclipse.  How could I pass up the opportunity, even though I’d be up to the wee hours of the night??  As hard as I tried, I just couldn't repress my giddy, childlike excitement.

As a photographer and astronomy enthusiast, it was an easy decision that viewing this phenomenon by eyes alone would not be enough.  I grabbed my Meade telescope, aligned it to North Star and immediately started tracking and viewing the Supermoon, which entirely filled my field of view.  As awesome as the sight was, that was just the prelude to the main event. 

Soon the eclipse began, and inch by inch, the ominous shadow of the earth advanced across the moon.  As the onslaught continued it left a wake of darkness and, as the night progressed, a secondary wake of blood red color, which is why this phenomenon is given the name “Blood Moon”.  I alternated between viewing the event through the telescope eyepiece and also doing some prime-focus astrophotography where my camera was directly mounted to the telescope, using it as a 2000mm prime lens.  The images I saw and captured were incredible and definitely worth the next day’s yawn-inducing effect.  Below is a series of photos across two and a half hours of the event. 

As I admired this lunar show, a thought hit me: wouldn’t it be cool to measure the phase of eclipse the moon was at?  A quick mental calculation told me this would be an easy task for BioTek Cytation Gen5 Image Analysis software, using Image Statistics with a Plug and a threshold.  But that could wait until morning, *yawn*.

Monday morning bright and early (brighter and earlier than I wished... due to my irresponsible late night gallivanting), I converted my photos into TIF files in ImageJ and opened them in Gen5 software.  First I measured the diameter of the moon using the software's Line Profile Tool, and defined an Image Statistics Plug that would perfectly outline and measure the moon. This would measure the precise size of the non-eclipsed moon which would be my reference.  Then I imported one of the photos with the eclipse. Using the Line Profile Tool again, I determined an upper threshold intensity value that would exclude the portion of the moon with high intensity that was not yet eclipsed (a value of 50,000 intensity was used). With a quick run of the analysis, I had my answer by using the Confluence measurement in Image Statistics.  90% of the moon was eclipsed in the photo I was analyzing. In the below screenshot, the blue in the image is what is excluded from the analysis, red is what is included as the portion of the moon that is eclipsed.  I quickly imported the other images and to my delight was able to measure the eclipse progression with ease! 

Now with all the easy image analysis figured out, we have 18 years until the next Blood Moon - plenty of time to develop a Cytation telescope module for an all-in-one astrophotography solution!   ... And plenty of time for me to recover from my lost sleep...

By: BioTek Instruments, Caleb Foster, Product Manager, Development

Thursday, October 1, 2015

Cytation Cell Imaging Multi-Mode Reader: Enabling Analysis of Phenotypic and Target-Based 3D Cellular Assays for Oncology Research

Cancer, the uncontrolled growth of abnormal cells in the body, is a general term used to account for more than 100 types of disease. According to Cancer Facts and Figures 2015, published by the American Cancer Society, nearly 600,000 U.S. residents will be lost to cancer by the end of this year. Global spending on cancer medications to combat each particular disease continues to increase, and for the first time crossed the $100 billion level in 2014. While concerted efforts to discover new anticancer drugs continue, this has not translated into high levels of success for potential new drugs, with up to 95% of candidate molecules failing clinical trials due to lack of efficacy or unforeseen safety concerns.

These shortcomings are in part the result of in vitro cell-based assay models that do not represent in vivo conditions. As an example, cells grown on two-dimensional (2D) hard plastic or glass substrates are easily prepared, but may not be representative of the true in vivo cell environment. Three-dimensional (3D) cell culture methods, in comparison, provide a matrix that encourages cells to organize into structures more indicative of the in vivo environment, thereby developing normal cell-cell and cell-ECM interactions in an in vitro environment.

Even as 3D cell culture methods bring about the hope of lowering lead molecule attrition rates, they also bring new challenges, particularly for assay readout systems such as conventional PMT-based detection and cellular microscopy. For example, cells aggregated into spheroid structures, which are much smaller than the area of the well of a 96-well microtiter plate are particularly challenging to monitor with conventional PMT-based detection used in microplate readers. However, in other 3D systems that encompass the whole microplate well such as scaffold-based 3D cell culture methods, PMT-based detection, which is designed to collect as much light as possible from a microplate well may provide better assay performance.

We invite you to read the Omics Tutorial, "Analysis of 3D Cell Culture Models: Enabling Phenotypic and Target-Based Assay of 3D Cellular Structures", in the September issue of Genetic Engineering & Biotechnology News to learn more how the combined automated digital widefield microscopy and conventional multi-mode microplate reading capabilities of BioTek's Cytation 3 and Cytation 5 offer a unique flexibility to enable a wide range of 3D cell culture studies.

Monday, August 31, 2015

How to turn your iPad into a cool 3D Holographic Projector for Fluorescence Microscopy

Hologram of a Cell undergoing mitosis
It's one of those concepts straight out of a science fiction movie like Star Trek - holographic projectors that show images and video in 3D.  But what if I told you that in only 3 simple steps, you could make your very own holographic imaging device to view your Fluorescence Microscopy images in vivid detail as shown above? Does it sound too good to be true??? Well, it's not! If you're interested in how to pull this off, and have an extra 30 minutes on hand for an easy DIY project, then read on! 
The concept of a hologram projector may sound quite complex, but using some simple optical principles (which I won’t get into in this blog), we can make one with the following supplies:
  • Tablet or cell phone (I tried it with both an iPhone and an iPad)
  • Clear plastic (you could use the clear plastic from a CD case cover)
  • Graph paper
  • Scissors
  • Box cutter knife
  • Tape
  • One of your coolest Fluorescence Microscopy images
  • Photo editing software 
Step 1: Cut 4 identical trapezoid shapes from the clear plastic. To do this, I cut out a basic trapezoid shape from my graph paper (shown below). I used that to trace the shape on my clear plastic, and then cut the four pieces with the box cutter. The size of the trapezoid below is for an iPad. I also made one for my iPhone where all the dimensions were half of what is shown below.   

Step 2: Tape the 4 clear plastic trapezoid pieces together as shown below.  That is actually all the hardware that you have to make.

Step 3: This step requires some knowledge of photo editing, so you may have to ask a friend or coworker for help.  The goal here is to turn your single cool fluorescent microscopy image into an image that contains 4 copies of the photo in progressive 90 degree orientations.  As an example, I took one of our high magnification Cytation 5 images that was acquired by BioTek’s Applications lab and converted it with Photoshop, as shown below.

Once the above image was created, I loaded it onto my iPad. 

Now that steps 1, 2, and 3 are done, all that is needed is to put everything together to use.

Place your iPad on a flat surface and load the image that you created in Photoshop onto your ipad.  Next, place the 4 sided clear plastic contraption (let’s call it the “holographic projector”) directly on top of the iPad in the center of the image with the small opening down, as shown below.

To best view the hologram, dim the lights in the room you are in and bend down so that you’re eyes are on the same level as the holographic projector.  What you should immediately see is a 3D-like projection of the cell that shows up in the center of the holographic projector (shown below).  Your first instinct very likely will be to try and touch the image, which both my 4-year old and I tried to do.  Incidentally, my 4 year old kept referring to the image as a "hot air balloon"; hopefully as a seasoned scientist I won't have to point out to you (as I did repeatedly to him...) that this is actually a cell undergoing mitosis, not a hot air balloon.  But I digress... Regardless of whether it’s a hot air balloon or a mitotic cell, it's still really cool to see it.

Below is one more example of an image viewed with this hologram projector. If you’ve gotten this far and want to have even more fun with this device, search on Youtube for “hologram” or “holographic” videos; there are numerous results of videos that can be used with this device that give you an even more spectacular viewing experience. However, sadly, there are no videos with fluorescently labeled cells (which I guess gives me opportunity for a follow up blog post). 

Now that all the hard work is done, all that’s left is to WOW your colleagues at your next lab meeting by using this to show off your microscopy images.  But please be forewarned: with a gadget like this, your coworkers may secretly think you are from the future so make sure any of your answers sound very futuristic too. Who says Science can’t be fun!?!

By: BioTek Instruments, Caleb Foster, Product Manager, Development

Tuesday, August 11, 2015

Moving Towards Gene Editing

For decades geneticists have theorized that targeted gene editing would provide an eloquent method to repair a defective gene, treat or prevent disease or allow selection of genetic traits in offspring. Regardless, successful efforts to incorporate targeted genetic material into higher organisms remain risky at best. Several approaches are currently being tested including the swapping of an abnormal gene for a good one, repairing a segment know to contain the deleterious error, or altering control of gene expression via editing of its transcriptional regulatory elements. All of the above approaches currently remain in the experimental mode.

One such example of gene therapy involves a mutated form of the gene RPE65 linked to an inherited disorder causing vision loss. In this case a harmless virus carrying the healthy gene was engineered as a vector to deliver the good gene1. While these studies have shown some success more importantly they point the way to future work.

Gene editing evolved in the early 2000s with the discovery of zinc finger nucleases and more highly evolved synthetic nucleases called TALENs. More recently a new technology is being adopted with high expectation of revolutionizing genome editing: CRISPR-Cas9 based genome editing. Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins were first discovered in bacteria and other prokaryotic organisms forming an immune system providing a form of acquired immunity to foreign genetic elements such as plasmids and phages. The CRISPR/Cas system has been adapted to gene editing in a wide variety of species by delivering the Cas9 protein and appropriate guide RNAs into the target cell. This approach allows the host genome to be cut at any desired target sequence location.

Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA:  http://dx.doi.org/10.1016/j.cell.2014.02.001
Currently in press, researchers are working to functionally correct Factor VIII gene chromosomal inversions in hemophilia A patients by using the CRISPR-Cas9 system and induced pluripotent stem cells (iPSCs). Proof of concept has shown rescue of factor VIII deficiency in a lethal mouse model2.  The hope is this technology will be transferrable to human disease targets in the near future.

1. Jacobson SG et al. “Improvement and decline in vision with gene therapy in childhood blindness.” New Engl J Med. DOI:10.1056/NEJMbr1412965

2. Park CY et al. “Functional Correction of Large Factor VIII Gene Chromosomal Inverstions in Hemophilia A Patient-Derived iPSCs Using CRISPR-Cas9.” Cell Stem Cell.  http://dx.doi.org/10.1016/j.stem.2015.07.001

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

Tuesday, August 4, 2015

Microscopy, Astronomy and Vincent Van Gogh

Anyone in biomedical research knows not all experiments work exactly like they are planned. This was the case a few weeks ago when I seeded some NIH3T3 cells that express GFP into the wells of a microplate. Maybe it was the recent images of Pluto transmitted from NASA's New Horizon's space probe or maybe it was the Van Gogh print of "The Starry Night" that used to hang in my daughter’s bedroom, but when I saw the completed montage image of NIH3T3-GFP cells that have been fixed and stained with DAPI my first thought was that it looked like stars and constellations.
Figure 1. DAPI stained NIH3T3 Cells Expressing GFP. Cells were fixed with 4% formaldehyde and then stained with DAPI nuclear stain. A total of 225 images in a 15 x 15 montage array using a 20X objective was rendered and stitched into a single image.  Scale bar represents 1000 ┬Ám.

Scientists examine things in particular ways using a combination of very sophisticated equipment, everyday instruments, and many unlikely tools. Some phenomena that scientists want to observe are so tiny that they need a microscope. Other things are so far away that a powerful telescope must be used in order to see them. What is fascinating to me is that despite the vast differences in size, things appear very similar. 
Figure 2.  Ultraviolet Coverage of the Hubble Space Telescope Ultra Deep Field. The Hubble Ultra Deep Field 2014 image is a composite of separate exposures taken in 2003 to 2012 with Hubble's Advanced Camera for Surveys and Wide Field Camera 3.

Despite the differences in true object size, astronomy and microscopy are very similar. Both of these fields of research use visual information as a means to maximize scientific expertise; yet the targets are often inaccessible to the human eye. Astronomy relies on telescopes to provide information about extraterrestrial objects, while microscopy utilizes microscopes to visualize cellular objects at much closer range. Even though the objects of astronomy are tremendously large, their distance from us renders them microscopic to the naked eye. At the most basic levels, both systems use much the same magnifier; essentially a tube with focusing lenses, but with markedly different focal lengths.

BioTek has a number of imager products that have produced some remarkable microscopic digital fluorescent and brightfield images. Coincidentally one of my colleagues in BioTek China created a 2015 calendar that shows the similarity between images generated by the BioTek Cytation readers and those taken by the Hubble Telescope.

The GFP expressing NIH3T3 cells that I plated were not evenly distributed and rather clumped making them unusable for the experiment that I had planned, but they certainly had the appearance of stars in the night sky. I have a pretty good idea as to why my cells are arranged the way they are, but astronomers have puzzled the same question about stars for centuries.

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