Tuesday, April 15, 2014

Cytation 3 - The First Year

The Cytation™ 3 Cell Imaging Multi-Mode Reader was launched one year ago today, and what an exciting year it’s been! Cytation 3 has circled the globe and been challenged by many interesting samples, ranging from cells, tissues to even whole organisms.

BioTek understands the importance of the cell as the most basic unit of life and its place as a model system for life science research. Our microplate instruments reflect this importance by enabling cell-based assays from seeding microplates with cells, automating assay workflows and detecting assay readouts, either kinetically or as endpoints. But it was the advent of the Cytation 3 that brought together conventional PMT-based microplate reading with CCD-based automated digital microscopy that allow for the capture of rich phenotypic information and meaningful quantitative data from the same microplate well. Our customers have used this enabling capability in the fight against cancer and neurodegenerative diseases, stem cell biology and with 3D cell culture systems in an effort to replace the use of animal models in drug discovery.

The response we received this first year has been amazing! Cytation 3 won the SelectScience Scientists’ Choice Award for Best Drug Discovery Product, the Miptec New Product Innovation Award, and the Thermo Fisher Scientific Extraordinary New Product Line Award. Additionally, Cytation 3 is a finalist in the Edison Awards competition - winners will be announced April 30, 2014.

There are now numerous Cytation 3 instruments in over 30 countries... far exceeding BioTek's initial forecast...and the momentum continues to build. Cytation 3 is helping researchers across the globe to "Think Possible" in research applications from angiogenesis to zebrafish embryo development, and everything in between.

4X Zebrafish Embryo

So as we celebrate Cytation 3's first anniversary, we wanted to thank all of our customers, employees, and supporters for making Cytation 3 a huge success. And just as Cytation 3 helps researchers to "Think Possible", we promise to continually "Think Possible" when it comes to developing new ways to increase and expand reading and imaging applications.


By: BioTek Instruments

Tuesday, March 25, 2014

You Can Bank on Stem Cells!

Stem cell research has remained in the spotlight for the better part of a decade now, for better or for worse. Since the discovery of human embryonic stem cells (hESCs) questions pertaining to their moral and ethical procurement and use for research and treatment has been scrutinized. Many of these issues were quelled by the discovery that somatic cells could be reprogrammed to a state similar to that of the hESC (i.e. pluripotent)  Most recently, Nature reported work surrounding a new methodology for reprogramming human somatic cells to a pluripotent state by chemically stressing them (likely many of us have a substantial reserve of them!) While intriguing, the report has come under scrutiny and independent verification of its usefulness as a method to generate induced pluripotent stem cells (iPSCs) remains to be seen. Regardless, the landscape is changing as reports on the use and interest in generating iPSCs with high genetic variability continues to infiltrate a broad range of scientific fields.

Early innovators in the field of stem cell biology pointed to the powerful experimental models that could be generated if easily harvested cells from an individual exhibiting a disease of interest could be reprogrammed to iPSCs.  By then differentiating these cells to a cell type particular to the disease being investigated it would be possible to examine a model with a more relevant genetic background. The use of iPSC derived cell lines would help overcome several disadvantages inherent with current models. These include primary human tissue or cadaveric tissue which have low availability and are subject to variability for a variety of reasons and immortalized cell lines that can exhibit significant alterations in biological function.  In fact many of the immortalized cell lines, such as CHO and HEK cell lines, were at the center of high-throughput screening and lead optimization campaigns over the recent past that have seen high candidate failure rates during clinical trials. It is thought that the use of cell lines with a more relevant genetic background coupled with phenotypic screening may prove more fruitful.

The increased efficiency of producing patient-specific iPSC lines over the past decade has lead to a relatively large number of cell lines derived from donors with a wide range of disease types.  As a mechanism to help provide worldwide access to the ever increasing number of available cell lines, efforts have been made to establish large banks to provide not only disease specific cells but genetically diverse iPSCs for population genetic studies. Several programs have now been sponsored in the US, UK, and China (http://www.nature.com/nbt/journal/v31/n10/fig_tab/nbt.2710_T2.html).

While the importance of such banks may be obvious the challenges are many. Significant work still needs to be done to standardize methods, characterize each cell line, improve reprogramming methods (particularly efficiency) and decrease costs. In fact cost may be the single biggest obstacle to the adoption of iPSC derived cell models by the pharmaceutical industry as the cost is estimated to be at least an order of magnitude more expensive than the use of immortalized cell lines. Without the backing of Pharma these banks may remain limited in scope if having to rely on public funds to finance future growth.

References

Novak, T.,  Grieshammer, U.,  Yaffe, M., and S. Madore (2012/14): Resetting the course of drug development: stem cell banking in support of drug discovery. In Drug Discovery World 15 (1), pp. 14–21.


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


Enabling the Latest Trends in Cell-Based Assays


BioTek Instruments focuses its attention on the latest scientific developments in cell-based assays. A central theme is to develop assays with greater physiological relevance such that disease models can be better understood, drugs developed with better efficacy and safety issues probed without resorting to extensive animal testing. This is manifest in the following trends seen over the last few years which are supplanting previous tools and methods.


BioTek microplate instrumentation is designed specifically to enable these latest trends in cell-based assays from both a workflow automation and detection perspective. The following application notes and scientific publications demonstrate this capability.

Primary Human Cells

Live Cell Kinetic Assays


Visit the Tech Resources section of www.biotek.com for more Application Notes covering our full line of microplate instrumentation.


By: BioTek Instruments

Friday, March 21, 2014

Using Raman Emission to Improve ELISA Reactions

The enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used assay technologies in the biomedical field today. The ELISA format typically uses a microplate to quantitate an analyte in a liquid sample through its interaction with a specific antibody. The technology combines the specificity of antibodies with a standardized assay process and format that is very amenable to automation. Because of its popularity, efforts are continually being made to improve assay performance. Toward that end, Sword Diagnostics has developed a substrate for ELISA reactions that produces a Raman emission that can be used as a direct replacement substrate for commonly used peroxidase conjugates.

Figure 1.  Energy Level Diagram of Raman and Fluorescence Signals.
Raman emission is a light scattering event where photons interact with the vibrational modes of a molecule’s electrons to gain or lose energy from the interaction and scatter at shifted frequencies (Figure 1). Unlike fluorescence there is no electronic transition and spontaneous emission of a photon. The Raman shift is dependent on the structure of the interacted target bonds and the environment of the bonds that the incident light interacted with. Resonance Raman is a special scattering event where the excitation wavelength is carefully tuned to be very close an electronic transition. Such overlap can result in scattering intensities which are increased significantly. The main difference between Raman scattering and fluorescence is the excited state lifetime. Fluorescence excited states are longer-lived than the 'virtual' states associated with Raman scattering.

Figure 2.  Comparison of Raman and Absorbance signal IL-6 ELISA Dose Response Curves. 
The data obtained using an absorbance-based TMB substrate and a Raman-based Sword peroxidase substrate both generate sigmoidal shaped calibration curves that can effectively be described using either a 4- or 5-parameter logistic fit (Figure2). However, the dose response of the Raman based reagents is significantly shifted leftward to lower concentrations. This indicates that the Raman substrate is capable of detecting approximately five-fold lower sample concentrations as compared to the absorbance based substrate with no other changes to the assay.

Raman and resonance Raman scattering measurements are usually performed using an expensive dedicated tunable laser based instrument with detection primarily taking place in the IR portion of the spectrum. Sword peroxidase chemistry employs reagents that have emissions in the near IR spectrum that an appropriately configured Synergy H4 Hybrid Multi-Mode Microplate Reader from BioTek Instruments is capable of detecting with high sensitivity. This combination of chemistry and hardware provides an increase in ELISA sensitivity without the additional costs of a dedicated instrument.


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

Thursday, February 20, 2014

Why Choose Cytation™ 3: Improving Data Generation Efficiency and Understanding from Cell-Based Assays


A large percentage of cell-based assays incorporate fluorescence or luminescence detection that are easily quantified using conventional microplate readers, even in higher throughputs. Quantitative data is rapidly generated, allowing users to make quick decisions; however, this may not always tell the entire story about what’s happening with the cells following treatment. This can lead to false assumptions and the wrong path forward in subsequent experimentation. In contrast, cell-based imaging assays allow visualization of events at the cellular level. However, image capture and storage can require considerable time and computer or network storage space.

This leads us to two reasons why someone would choose our Cytation™ 3 Cell Imaging Multi-Mode Reader instead of a separate imager and microplate reader when performing cell-based assays: confirming cellular activity and hit-picking. We performed two experiments to illustrate these benefits.

Cellular Imaging Confirmation

To illustrate the importance of confirming cellular activity, we repeatedly dosed hepatocytes with a drug over multiple days and assessed cell viability/toxicity. This is very similar to hepatotoxicity studies performed in the drug discovery process following lead molecule generation. Cryopreserved human hepatocytes (BioreclamationIVT, Baltimore, MD) were cultured in 96-well collagen-coated plates at a concentration of 50,000 cells/well. Several concentrations of the known cytotoxin, camptothecin, were added to the cultures on a daily basis over a seven-day period. Potential cytotoxicity from the compound was measured at 1, 3, and 7 days following treatment initiation. Total and cytotoxic cell numbers were assessed using the Hoechst 33342 cell permeable nuclear stain, and CellTox™ Green Cytotoxicity Assay from Promega Corporation (Madison, WI), respectively. All measurements were performed using Cytation 3.

By looking at results from the mean green fluorescence using the CellTox Green dye (Figure 1), it is apparent that fluorescent values increase in a dose- and time-dependent fashion for days 1 and 3, indicating that the cytotoxic effect from the compound is also increasing in the same manner. However on day 7, cytotoxicity increases rapidly at lower camptothecin doses, but then falls off to levels below the uninhibited control at higher concentrations. If one only had access to this data set generated by a microplate reader, it would be difficult to assess exactly what effect the higher camptothecin doses have on hepatocytes after a seven-day dosing. However, as we show next, by imaging the well, it then becomes clear as to why the fluorescence is “disappearing” from these wells.

The captured 4x images of hepatocytes treated with 20,000 nM camptothecin (Figure 2) illustrate how cytotoxicity increases from day 1 to day 3. Total cells show up as blue, while dead cells are seen as green. After a seven day dosing, though, the camptothecin concentration is completely toxic to the cells, causing them to detach from the bottom of the well and float up into the media. Only by incorporating imaging would this phenomenon be noticed, allowing for the correct conclusion to be drawn about this compound.

Figure 1.  Hepatotoxicity results following 1, 3, and 7 day camptothecin treatments.
Figure 1.  Hepatotoxicity results following 1, 3, and 7 day camptothecin treatments. 

Figure 2. 4x images of hepatocytes treated with 20 µM camptothecin showing live (blue) and dead (green) cells
Figure 2. 4x images of hepatocytes treated with 20 µM camptothecin showing live (blue) and dead (green) cells.

Hit Picking

In high content phenotypic assays, every microplate well is imaged, even though only a few wells may ultimately contain data of interest. This takes a great deal of time, and requires lots of data storage. Cytation 3 may be programmed with an intensity threshold that triggers the instrument to image only those wells that meet, or “hit”, a user-programmed criterion when the plate is read instead of the entire plate. This hit picking dramatically reduces analysis time and data storage requirements for greater efficiencies.

To illustrate the “hit picking” process, we performed an experiment to identify potential cellular hypoxia inhibitors. Immortalized keratinocytes were cultured in the presence of cobalt chloride (CoCl2), a known inducer of hypoxia-like responses, along with a REDOX compound library (Enzo Life Sciences, Farmingdale, NY) made up of several known antioxidants. Hoechst 33342 cell permeable nuclear stain and fluorescence-based Cyto-ID® Hypoxia/Oxidative Stress Detection Kit from Enzo Life Sciences were used to identify cell numbers and assess inhibition of oxidative stress and hypoxia. Prior to reading the assayed microplates, a single Cytation 3 Hit Pick protocol was created. The protocol used the results from a monochromator-based read of the signal from the hypoxia dye to initiate imaging of wells whose RFU values were greater than one standard deviation lower than the average of four positive control wells. This corresponded to greater than or equal to 50% inhibition of the hypoxic condition.

Per Figure 3, a total of 27 wells were imaged using the protocol, including 15 "hit picked" wells and 12 control wells in row H. The time to capture three images from these wells is 3 minutes and 22 seconds. When compared to the total time required to capture three images from all 96 wells, which is 12 minutes, this represents a 3.5 fold savings in time, as well as required storage space.

Figure 3.  Selected wells for imaging using “Hit Pick” protocol criteria.
Figure 3.  Selected wells for imaging using “Hit Pick” protocol criteria.

Summary

Cell-based microplate reader and imaging-based assays play an essential role in current life science research. While both yield important information necessary for making critical decisions, each can suffer from crucial shortfalls when performed on an instrument only capable of performing one type of detection.  The Cytation 3’s combined capabilities, however, alleviate these concerns by providing an instrument which can streamline processes and ensure that all relevant experimental data is available during the decision making process.


Want to learn more? Check out these pages:

By: BioTek Instruments, Brad Larson, Principal Scientist

Friday, February 7, 2014

Comparison of 3D Cell Culture Technologies to Plated Cells in 2D for Oncology and Toxicology Applications


A central focus for improving drug efficacy in clinical trials over the last decade has been to increase the biological relevance of assays performed early in the drug discovery process. Yet it remains difficult to simulate an in vivo response to drug using an in vitro assay, where the cells are grown on hard plastic or glass substrates, in a two-dimensional (2D) format which is not representative of the in vivo cellular environment. When examining cells within a tissue, it can be observed that cells interact with neighboring cells, and with the extracellular matrix (ECM) to form a communication network. This communication controls a number of cellular processes including proliferation, migration, and apoptosis. However, most of the tissue-specific architecture, cell-cell communication, and cues are lost when cells are grown in a more simplified 2D manner. Therefore, more advanced cell culture methods are required to better mimic cellular function within living tissue.

3D cell culture serves to meet this demand by providing a matrix that encourages cells to reorganize into a structure more indicative of an in vivo environment; thereby allowing normal cell-cell and cell-ECM interactions to develop in an in vitro environment.

In an upcoming webinar to be presented on February 27, we will describe two common formats for culturing cells into 3D. These include the RAFT™ collagen-based 3D cell culture system from TAP Biosystems (Hertfordshire, UK) and GravityPLUS™ platform from InSphero (Schlieren, Switzerland). Individual project results will demonstrate how these 3D cell culturing procedures can be automated using the non-contact dispensing capabilities of the MultiFlo™ FX, and microplate reading and imaging-based assays can be performed using the Cytation™ 3 to generate more relevant data for oncology and toxicology studies than traditional 2D methods.

Register for the webinar to learn more.


By: BioTek Instruments, Brad Larson, Principal Scientist

Monday, February 3, 2014

What Would You Do With a Free Cytation 3?

There are a lot of ways that BioTek’s Cytation 3 Cell Imaging Multi-Mode Reader has already made a splash in the market. From awards, to price point, to applications to BioTek giving away a free one…. wait?!?! Giving away a free one, you say?!? That's right, you heard me correctly; BioTek is actually giving away a free Cytation 3 to one lucky winner. How is this possible? The short answer: because we "Think Possible". We're so excited at how Cytation 3 is revolutionizing research that we are holding a Think Possible Application Contest.


The contest requires entrants to describe an application that they think is possible for their specific research using the Cytation 3.  The theme of this contest is Cytation 3 turning improbable, even previously considered impossible applications into Possible, and we want to share the love.  Alas… the only eligibility exclusion for the contest - you cannot be a BioTek employee or distributor, so I’m out of luck (wiping away a tear from my cheek).  However, I still had plenty of fun imagining what Cytation 3 and I would accomplish together if I won.

When I first heard about this contest, I went home and began to dream of what unusual or even crazy applications I could do if I won my very own Cytation 3. As I sat on my couch, shortly, it hit me… this contest is a chance for one lucky scientist to make scientific history. I mean, how often do you as a scientist get the chance to go beyond the limited confines of your grants and budgets and dream of what you could accomplish if given the right resources? For FREE. Use your imagination. What would you do? Think of the fun you could have with the Cytation 3, the impact on your area of research, the new and innovative ideas you could turn into reality.  

Now it’s your turn. Here’s the challenge - what will you do if you win?  How will you think outside the box? How will you surmount the barriers you’ve encountered in your research? Take a few moments to enter the contest - let us know! We LOVE to hear your ideas, and we are looking forward to enabling one lucky scientist. It could be you. Because when you Think Possible, you will see the world in a whole new way!


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