Applications Thursday – GPCR Activation via cAMP or Inositol Phosphate Production is Measured in Live Cells Using HTRF® Chemistry

GPCRs have two major signaling pathways - the regulation of cAMP levels and the increase in intracellular Ca2+ triggered by inositol 1, 4, 5- triphosphate (IP3). Specific G proteins activates these signaling pathways and they are associated with different receptors. Activation of a Gs or Gi coupled receptor results in the increase or decrease of cAMP levels, respectively. While activation of a Gq coupled receptor activates phospholipase C (PLC) and triggers the inositol phosphate (IP) cascade.

Cisbio Bioassays has assay kits able to measure the activation of Gs, Gi and Gq coupled receptors in one assay, using their new generation Lumi4-Tb™ TR-FRET Cryptate. This chemistry allows for the detection of two signals in one well using a green acceptor (λ = 520 nm) and a red acceptor (λ = 665 nm). This same chemistry is used in Cisbio’s Tag-lite® technology.

IP-One and cAMP experiments were performed on the PHERAstar HTS microplate readers using Simultaneous Dual Emission detection (detailed in Application note 209 . With this unique feature, the plate is read only once for dual emission assays, thereby decreasing time and variability. All Cisbio chemistry can be performed on the PHERAstar FS, which uses a UV Laser for excitation. The UV Laser will allow for greater sensitivity and faster read times (see Application Note 222 for IP-One data using the UV Laser) . The POLARstar and FLUOstar Omega with advanced assay technology also perform this chemistry, but in a non-HTS fashion.

Fun Fact: One Week Left to Vote for SLAS Board of Directors

The Society for Laboratory Automation and Screening (SLAS) is electing three new members to serve for three years on the SLAS Board of Directors and there is only one week left to vote. Amongst the 8 candidates is Dr. E.J. Dell, the International Marketing Manager for BMG LABTECH. Dr. Dell has attended both ALA and SBS meetings over the last six years while with BMG LABTECH and he hopes to bring some fresh ideas to SLAS if chosen. Some of those ideas include:

• Further miniaturization to smaller volumes – This will not only save time and money, but assays will also be performed in a more realistic lower volume environment 
• Increased presence of core-facilities at SLAS – Core facilities that do specialized screening have changed the landscape of HTS 
• Broadening automation and screening to other scientific fields like Toxicology and Environmental Biology – This would encourage those fields of study to migrate away from whole animal bioassays

If you are a member of SLAS and have not yet voted, you may do so here: https://www.slas.org/election/index-2013.cfm?goback=%2Egmp_3363923%2Egde_3363923_member_189718058.

See you in January at Booth #721 at SLAS2013 in Orlando!

Did you know that BMG LABTECH is presenting a webinar tomorrow, November 28th at 12:00 EST on Cell-Based Microplate Assays?

In collaboration with Drug Discovery and Development, BMG LABTECH is hosting a free webinar entitled Developing and Optimizing Cell-Based Microplate Assays. In this webinar, three different speakers will highlight different aspects of developing cell-based assays in a microplate format. They include:

• Optimizing Measurements for Microplate Cell-based Assays
  Catherine Wark, BMG LABTECH, UK
• Miniaturization and Automation of HTRF® Cell-based Assays with the Echo® Liquid Handler
  Bonnie Edwards, Labcyte, Inc., USA
• Identification and characterization of allosteric modulators of GPCRs using an HTRF® cellular assay
  Jeff Jerman, PhD, MRC Technology, UK

Sign up here to join us at 12:00 EST tomorrow for this live webinar, panelists will be available for questions. Reproductions of the webinar will be available shortly upon request.

FAQ: What are the advantages of well scanning?

Under normal reading conditions, most plate readers take a single measurement in the center of each well. For cell based assays where the cell distribution is uneven a single center well reading is insufficient. In some cases, a single center point reading may completely miss the material of interest in a large well format.

To overcome this problem, BMG LABTECH has added an advanced Well Scanning feature to its plate reader lines. In Well Scanning mode the microplate reader can take multiple measurements in each well with up to 30 x 30 data points. Well scanning (30 x 30 matrix) and Direct Optic Bottom reading provide the PHERAstar FS with an excellent platform for advanced high resolution cell-based analysis in 384 well plates. This enables the PHERAstar FS to perform cell-based assays that other microplate readers cannot.

Focus On: Developing and Optimizing Cell-based Microplate Assays Webinar

On Wednesday November 28th at 12:00 EST, Drug Discovery and Development will host a webinar that details how to develop and optimize cell-based assays in a microplate format.

Many researchers need to migrate a biochemical assay to a cell-based format to get more information, or they need to increase their throughput of their current cell-based assay. One way to achieve this is to create a cell-based assay in a microplate format. Over the last several years many new cell-based assays have been developed, in addition, there have been improvements in instrumentation that are needed to measure and dispense these cell-based assays. Join this webinar to hear three presenters discuss three different aspects of cell-based microplate assays:

• Optimizing Measurements for Microplate Cell-based Assays
  Catherine Wark, BMG LABTECH, UK
• Miniaturization and Automation of HTRF® Cell-based Assays with the Echo® Liquid Handler
  Bonnie Edwards, Labcyte, Inc., USA
• Identification and characterization of allosteric modulators of GPCRs using an HTRF® cellular assay
  Jeff Jerman, PhD, MRC Technology, UK

For more information please visit our webinar sign-up page. The webinar is free to join so sign up now!

Applications Thursday – Dual Glow Reporter Gene Assays Benefit from Dual Emission Detection and Longer Bandwidth Filters

A new second wave of dual glow luciferase reporter gene assays are now available from various companies (Pierce, Promega, Life Technologies). Other than Firefly and Renilla, there are now Gaussia, Cypridina, Green Renilla, and Red Firefly luciferases that are available and that can be multiplexed together, or even with fluorescent signals.

One main difference between these new dual glow reporter assays  and earlier dual glow assays like DLR™, is that the two different luminescent signals can be differentiated at different wavelengths, rather than at different time points. This enables higher throughput because there is no need for injection and no need for a minimal measurement time, the only limitation is in the instrumentation.

Larger bandwidth filters can capture more signal. 

Two features on instruments that will benefit these dual glow assays are Dual Emission Detection, and Longer Bandwidth Filters. Since two emission wavelengths need to be measured per well, microplate readers with Dual Emission Detection, like the POLARstar Omega and PHERAstar FS, will measure these assays in half the time compared to instruments that do not have Dual Emission Detection. Dual Emission Detection also helps to correct with read-to-read variations that can occur with reading the plate twice.

In addition, since these luminescent signals are not as bright as fluorescent signals, it benefits from collecting a larger area of the emission signal at each wavelength. Thus microplate readers with the ability to measure broad bandwidths (50-100 nm), like the POLARstar Omega and PHERAstar FS because they use longer bandwidth filters, will be more sensitive in these assays.

Learn more about it here, Wavelength Based Dual Glow Reporter Genes.

Fun Fact – Longer Telomeres, Mean Longer Life Expectancy

A new 20 year study published in Molecular Ecology shows that telomere length can be used to predict the lifespan of Seychelles Warblers, which are birds contained on an island and that have no natural predator. This study measured telomeres, which are end caps of chromosomal DNA that get shorter each time DNA is copied. Across the lifespan of these warblers, the study found that having shorter telomeres at any age is associated with an increased risk of death. The study showed that “short and rapidly shortening telomeres were a good indication that the bird would die within a year.” Since telomeres can be attacked by oxidants, it is thought that stress may play a role in telomere shortening. Though this is just one study, it seems that telomere length is a better indicator of biological age and future life-expectancy than real age.

For more information click here: http://www.sciencedaily.com/releases/2012/11/121119213144.htm

Did you know that a BMG LABTECH microplate reader can take some of the workload off of your High Content Screening (HCS) system?

High content screening allows researchers to see the phenotype of a cell and how it is affected by various treatments. Information about cell shape or where two fluorescent proteins co-localize isn’t information that can be obtained many other ways. However, some changes—cell motility, cell death, fluorescent protein expression levels—might require more information than a standard plate reader can provide but don’t require the full imaging capability of an HCS system.

Readers like BMG LABTECH’s PHERAstar FS can focus precisely from the bottom of the well and measure cell-based assays in scanning mode, taking hundreds of data points per well to create a fluorescence intensity map. It isn’t possible to see what’s happening in individual cells, but it’s easy to see cell coverage and uniformity, or where in the well expression is happening. An assay that can take two hours on an HCS system would take only eight minutes on the PHERAstar FS, meaning that researchers can collect more data per hour and that expensive HCS equipment is free to be used for methods that truly require its capabilities.

This figure shows the well scanning capabilities of the PHERAstar FS
using a 384-well plate seeded with cells expressing different levels of GFP.

 Here is an application note that describes in detail how the PHERAstar FS outperforms a leading CCD device in HTRF® mode:
HTS Instrument Discovers Low Affinity Inhibitors of the Inositol Phosphate (IP) Signaling Pathway.

For more information on Developing and Optimizing Cell-based Microplate Assays, join us for our webinar on Wednesday, November 28. More information can be found here:
http://www.bmglabtech.com/webinar/cell-based-assays.cfm

FAQ: How can cell migration or invasion assays be measured on a microplate reader?

Cell migration and cell invasion are important parameters to investigate for different cell types and for different disease states. For instance, tumor cells undergo a transformation where they metastasize to another area of the body by invading different tissues. Or leukocytes need to migrate to different areas of the body during infection. Therefore, assays that can measure cell migration and cell invasion are important tools to have for a microplate reader.

One way to measure cell invasion is to use the FluoroBlokTM assay from BD. Well chambers with a membrane are placed into a 96 well microplate and fluorescently labeled cells are seeded on top of the membrane. Measurements are taken from the microplate bottom to measure how quickly the fluorescently labeled cells invade through the membrane. Review this application note to learn more: Analysis of Prostate Tumour Cell Invasion Using BD FluoroBlok™and FLUOstar OPTIMA

Another way to measure cell migration is to use the Platypus® OrisTM Cell Migration Assay. Here a plug is placed in the center of a microplate well and fluorescently labeled cells are seeded on the outside of the plug. The plug is the removed and measurements are taken to watch how quickly the migration of the cells occurs toward the middle of the well. Review this application note to learn more: Analysis of migration using the Oris(TM) Cell Migration Assay-TriCoated kit on the POLARstar Omega.

Focus On Histone Deacetylases (HDACs)

As the field of epigenetics unfolds, various enzymes have now become important targets for drug screening as cures to various diseases, such as cancer. One such enzyme family is the Histone Deacetylases (HDACs), which modify histones and which in turn helps to regulate gene transcription. Though the mechanism of action is not fully known, it has been shown that inhibition of HDACs results in the overacetylation of histones that in turn can lead to a controlled cell death or apoptosis. As such, several HDAC inhibitors (HDIs) are in phase I or II clinical trials, for example suberoylanilide hydroxamic acid (SAHA; ZOLINZA®, Merck).

Check out one of our POLARstar application notes that describes an HDAC assay: A Fluorescence Based Assay of the Epigenetic Enzyme Histonedeacetylase 1 (HDAC1)

Inside Applications: Methyltransferase, Acetyltransferase, Kinases, and GTPases

Methyltransferase, Acetyltransferase, Kinases, and GTPases Can All Be Measured with Transcreener® Assays

• The universal Transcreener® assays measure the byproducts of common biological enzymatic reactions
• For epigenetic studies, AMP/GMP can be monitored for methyltranferase or acetyltransferase activity
• ADP for kinases, ATPases, or helicases; GDP for Gproteins; and UDP for glycosyltransferases

This application note demonstrates the validation of the BMG LABTECH PHERAstar Plus and PHERAstar FS instruments for use with Transcreener FP assays in 384-well format. BellBrook Labs offers four competitive immunoassays for direct detection of ADP, AMP/GMP, UDP and GDP. These nucleotides are often byproducts in enzymatic reactions and their quantification allows calculation of enzymatic activity.

Transcreener is a registered trademark of BellBrook Labs.

Fun Fact: Right now, Epigenetic is one of the fastest growing fields of life science.

The field of Epigenetics has risen in popularity over years, as such so has the number of publications with the term Epigenetic in the title or abstract. Starting in 1990, the year and the term Epigenetic was searched on Pubmed  to see the number of results that were returned. Though this graph is a crude estimation, if nothing else it shows the growing trend for using the term epigenetics in research publications. It is compared to the term Proteomics which also had a rise in use but seemed to peak earlier in the decade. Will Epigenetics also peak ~1000 publications/year?

Did you know about the different epigenetic assays that can be performed on a microplate reader?

Multimode microplate readers have become a necessary tool in the modern life science laboratory and as a consequence many biochemical and cellular assays are made specifically for a microplate format. With research into epigenetics exploding over the last couple of years, the number of assays available for microplate readers has also grown.

Here are some different companies that offere assays that can all be performed on a BMG LABTCH microplate reader:

• Promega 
• CISBIO
• BellBrook Labs 
• Life Technologies
• Epigentek
• Qiagen
• Cayman Chemical

FAQ: What is epigenetics and why is it such an exciting field of biology right now?

Epigenetics  is the study of physical changes to the genome that influence gene expression and that can lead to inheritable changes in cells. Basically epigenetics put to rest the argument of nature vs. nurture, it is both.

Specifically epigenetics encompass three changes to the genome:

• DNA methylation is thought to be involved in silencing regions of DNA so they cannot be transcribed
• Histone modification (i.e. acetylation, methylation, ubiquitylation, phosphorylation and sumoylation) can lead to histone remodeling, possibly making it easier for transcription 
• Small Interfering RNA (siRNA) are small fragments of RNA (<30 nucleotides) which can bind to and regulate different promoter regions of DNA. 

The study of epigenetics has led to important understandings of  genetic differences, such as why one identical twin can contract a disease such as lupus, while the other one does not. In addition, epigenetics is allowing us to change the way we view and treat some cancers such as squamous cell carcinoma and liver cancer.

As a consequence new assays are being developed all of the time to study epigenetics. Revisit our Blog in the coming week as we will investigate some of those assays that can be performed on a microplate reader.

Friday Focus: Luciferase Reporter Assays

Reporter genes like luciferase are one of the most fundamental and useful tools in a molecular biologist’s tool box. As popular and as useful as something like Firefly Luciferase is, it isn’t ideal for every application. At 61kDa, it’s large enough that as a fusion protein, it may interfere with some of the processes it’s intended to track. Luciferase substrate, D-luciferin, produces detectable levels of background luminescence that interfere with detecting biologically relevant signals from the reporter itself. And any luciferase reporter would be more useful if it could be detected at lower levels.

Promega’s new NanoLuc LuciferaseTM reporter (isolated from a deep sea shrimp) is an enhanced tool for researchers. It’s more than three times smaller than Firefly Luciferase, so it’s less likely to interfere with biological processes. Nanoluc’s substrate produces lower levels of background luminescence than traditional luciferase assays and the NanLuc enzyme itself emits light about 100x brighter than Firefly. This means that researchers are more likely to see low levels of activity and also design assays that work at biologically relevant concentrations. Thousands of BMG LABTECH customers use Promega’s reporter genes on our microplate readers  daily and we know they are excited to try the newest one out too.

Applications Thursday – Chroma-Glo Assay in 1536-well Microplates

Miniaturizing and AutomatingCell Viability and Reporter Assays for High-Throughput and Ultrahigh-ThroughputScreening

Screening facilities require simple solutions for measuring and correlating a variety of parameters of cellular processes. Robust assays and high-precision, low-volume, non-contact pipetting systems are now providing this capability. This application highlights the scalability of the CellTiter-Glo®, Caspase-Glo™ 3/7, and Chroma-Luc™/Chroma-Glo™ Technologies for high-throughput and ultrahigh-throughput cell-based screening in low-volume 384- and 1536-well formats.

For more information click here. 

Fun Fact: Movember

According to uk.movember.com:
"Movember, the month formerly known as November, is when brave and selfless men around the world grow a moustache, with the support of the women in their lives, to raise awareness and funds for men’s health - specifically prostate and testicular cancer.

Movember was established in 2003 by a few friends over a beer in a pub just outside Melbourne, Australia. The goal was simple – to create a campaign promoting the growth of the moustache among likeminded people and having fun along the way. It is about real men, talking about real issues and changing the face of men’s health, one moustache at a time. Movember now spans the globe, with campaigns in 21 countries in 2012."

Four members of the BMG LABTECH UK team are proud to be paritcipating and raising money and awareness for men's health issues.

Check up on the BMG LABTECH team including:

Robert Mount
Martin Lane
Lee Archer
Martin Fisher

and help us raise money and awareness for this cause.

For more information visit: http://uk.movember.com/

Did you know that you no longer need injectors to perform dual reporter assays using luciferase?

Stable and bright luminescence reporters like red-luciferase, green-Renilla, Gaussia and Cypridina lets users measure two luciferase constructs at once using a long-lived luminescence suitable for liquid handling. Traditional dual reporter assays, like dual luciferase, use time and two different injections to allow users to discriminate between both reporters. Glow-type dual reporter assays use two luciferase constructs that each emit a light at a different wavelength, meaning that a luminometer capable of accepting filters, like any of BMG LABTECH’s Omega microplate readers, can discriminate between two different luciferase enzymes emitting at the same time.

For more information about some of these newer luminescence assays please see Pierce or Promega.

Focus On BMG LABTECH Customers

For over 20 years BMG LABTECH has been providing quality instrumentation for life science labs, core facilities, and high-throughput screening centers. As such we have tens of thousands of satisfied customers. If you are looking for a new microplate reader and want an unbiased opinion, read and listen to what our customers have to say about BMG LABTECH products and service. German engineering is just the beginning, satisfied customers are the result.

"The Cancer Research UK Drug Discovery Unit at the Paterson Institute for Cancer Research has recently purchased a PHERAstar FS multimode plate reader. I had previously used this plate reader and worked with BMG over a number of years.  I have always been very impressed with the quality of data produced by the PHERAstar across various applications. The customer service, from purchase right through to downstream support, has always been prompt, friendly and incredibly helpful. Our lab was in need of a top spec plate reader that would produce the highest quality data for our assay scientists yet remain easy to use and approachable for occasional users. When demonstrated alongside other top spec plate readers, the PHERAstar outperformed on each level, with the  most marked improvements seen when measuring europium-based TR-FRET."
Alexandra Boakes
University of Manchester, UK
The Paterson Institute for Cancer Research, Drug Discovery Unit

Read more customer testimonials here: http://www.bmglabtech.com/testimonials/testimonials.cfm.

Applications Thursday – SNP Genotyping

Single Nucleotide Polymorphisms (SNPs) can be used as genetic markers in Agrigenomics to identify seeds or plants in breeding programs. There are several different chemistries that can perform SNP Genotyping amongst which are KASPar®, Taqman®, and Invader®. These chemistries rely on the detection of multiple fluorophores for each sample and when screening thousands of samples an instrument is needed that can reliably read these multiple fluorophores in a high-throughput manner.

 The PHERAstar Plus and the PHERAstar FS are the perfect instruments to handle these screens. Having Dual Emission Detection and the ability to read 1536-well microplates in less than a minute, the PHERAstar microplate readers make SNP Genotyping a snap.

For more information, check out this application note showing how the misread rate of SNP Genotyping using KASPar® is less than 0.1, while a recent publication shows how the PHERAstar and KASPar® have been used for the genetic mapping of Pigeonpea and Legumes.