ProbesOnline™ August 2013—Onstage incubator for time-lapse imaging, chemiluminescence ELISAs, oxidative stress detection
In this issue
FEATURED NEW PRODUCTS
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Keep your cells healthy during imaging, and see the results in time lapse—EVOS® Onstage Incubator for EVOS® FL Auto Imaging System | |
Developing sensitive chemiluminescence ELISAs—DynaLight™ Substrate with RapidGlow™ Enhancer | ||
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ABfinity™ recombinant antibodies—New antibodies for smooth muscle actin | |
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Accurate microRNA quantitation—MyQubit miRNA Assay | |
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Antibody conjugates for the detection of CD33—New anti–human CD33 conjugates for flow cytometry |
NEW APPLICATIONS
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Enzyme-mediated methodology for the site-specific radiolabeling of antibodies based on catalyst-free click chemistry |
PROVEN PERFORMERS
PRODUCT OFFERS
Discounts and promotions
Stretch your lab budget with our discounts and promotions
Life in the Lab magazine
Find product information, science news articles, special offers, and more
DEPARTMENTS
On the web
Model systems for Parkinson’s disease, and
flow cytometric assays for apoptosis
Imaging corner
Reducing endogenous background with the Endogenous Biotin-Blocking Kit
Highlight from BioProbes® Journal
Live-cell staining of pluripotent stem cells: Alkaline Phosphatase Live Stain for early identification of induced pluripotent stem cells
Molecular Probes® webinar series
Fluorophore selection, and
Cell proliferation for flow cytometry
UPCOMING EVENTS
6th Annual Great Plains Analytical Cytometry Association (GPACA)
September 13
University of Iowa, Iowa City, Iowa, USA
Great Lakes International Imaging and Flow Cytometry Association (GLIIFCA) 2013
September 27–29
Renaissance Center Marriott, Detroit, Michigan, USA
Ohio River Valley Cytometry Association Annual Meeting
September 18
Cincinnati Children’s Hospital, Cincinnati, Ohio, USA
OTHER PUBLICATIONS
BioProbes® Journal of Cell Biology Applications
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The Molecular Probes® Handbook
FEATURED NEW PRODUCTS
EVOS® Onstage Incubator for EVOS® FL Auto Imaging System
What it is
The EVOS® Onstage Incubator is an environmental chamber designed specifically for the EVOS® FL Auto Imaging System. The environmental chamber fits on the automated X-Y stage. A small, separate control unit supplies the power and gas (air, CO2, and N2 for hypoxia experiments), and controls humidity and temperature. Several interchangeable vessel holders are available and make time-lapse imaging easy and convenient.
What it offers
- Easily controls a fully integrated environmental chamber for live-cell time-lapse imaging
- Precisely maintains physiological or nonphysiological conditions
- Intuitively sets all environmental and image acquisition parameters from the EVOS® FL Auto interface
How it works
Together, the EVOS® FL Auto system and the EVOS® Onstage Incubator enable precise control of temperature, humidity, and three gases for time-lapse imaging of live cells under both physiological and nonphysiological (e.g., hypoxia) conditions. Environmental settings and image acquisition parameters are all seamlessly integrated into the EVOS® FL Auto interface, creating a high-performance inverted imaging system with unmatched flexibility, ease of use, and superb optical performance for demanding live-cell time-lapse imaging experiments.
- Learn more about the EVOS® Onstage Incubator
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Product | Quantity | Cat. No. |
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EVOS® Onstage Incubator | 1 each | AMC1000 |
EVOS® FL Auto Imaging System | 1 each | AMAFD1000 |
FLoid® Cell Imaging Station rides the BioBus to inspire love of science
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BioBus, a high-tech lab on wheels, provides K-12 students authentic lab experiences through hands-on lessons in cell biology. With the help of sponsors and supporters such as Life Technologies, the BioBus has brought interactive science education to more than 20,000 students every year. In support of this effort, Life Technologies loaned a FLoid® Cell Imaging Station to travel aboard the BioBus. |
DynaLight™ Substrate with RapidGlow™ Enhancer
What it is
DynaLight™ Substrate with RapidGlow™ Enhancer is a chemiluminescence detection reagent used in microplate ELISAs and automated immunoassays. This product provides superior sensitivity and dynamic range compared to colorimetric methods, when developing assays.
What it offers
- Sensitivity and dynamic range—pg/mL detection, with up to 5 logs of dynamic range
- Flexibility—chemiluminescence can be read minutes or hours after addition
- Stability—signal level remains consistent for over 1 year when stored refrigerated
How it works
In an ELISA, DynaLight™ Substrate with RapidGlow™ Enhancer is added to a bead system or microplate well that has been washed to remove any unbound reagents. Bound alkaline phosphatase conjugate interacts with the DynaLight™ Substrate, resulting in a high-energy intermediate compound that emits the energy as light when it decays. Light output rapidly ramps in minutes to a steady signal. This enzyme-triggered chemiluminescence provides pg/mL sensitivity with up to 5 logs of dynamic range.
- Learn more about DynaLight™ Substrate with RapidGlow™ Enhancer
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Product | Quantity | Cat. No. |
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DynaLight™ Substrate with RapidGlow™ Enhancer | 100 mL | 4475406 |
DynaLight™ Substrate with RapidGlow™ Enhancer | 1 L | 4475410 |
DynaLight™ Trigger Solution | 100 mL | 4475403 |
DynaLight™ Trigger Solution | 1 L | 4475409 |
New antibodies for smooth muscle actin
What they are
ABfinity™ recombinant monoclonal and oligoclonal antibodies offer consistent results, minimizing the need to revalidate working antibody dilutions for your experiments each time you order. Life Technologies currently offers hundreds of ABfinity™ recombinant antibodies, and we are actively developing more.
We have 2 new antibodies to smooth muscle actin. Smooth muscle is found in the walls of blood vessels, lymphatic vessels, and a number of other tissues, where it provides mechanical support and is a major component of the contractile apparatus of those tissues. One of the primary components of smooth muscle is actin, which has 6 known isotypes in mammals. Smooth muscle actin exists in α- and γ-actin isoforms, with the α isoform prevalent within vascular smooth muscle cells.
What they offer
- Specificity—undergo rigorous validation
- High performance—proven consistency from lot to lot
- Efficiencyt—detect low-level targets with a small sample
How they work
ABfinity™ antibodies are produced by transfecting mammalian cells with high-level expression vectors containing immunogen-specific heavy- and light-chain rabbit antibody cDNA. This highly reproducible process results in superb consistency in lot-to-lot antibody performance.
ABfinity™ oligoclonal antibodies are mixtures of recombinant monoclonal antibodies. These combine the improved signal strength that can come from using polyclonal antibodies, with the highly reproducible results you get from ABfinity™ monoclonal antibodies.
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Immunohistochemistry analysis of human uterine leiomyoma tissue section probed with ABfinity™ smooth muscle actin recombinant rabbit oligoclonal antibody. Horseradish peroxidase (HRP)-conjugated goat anti–rabbit IgG was used as secondary antibody. Diaminobenzidine (DAB) was used to reveal the HRP, and hematoxylin was used to stain the nucleus. |
Product | Quantity | Cat. No. |
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Smooth Muscle Actin Oligoclonal Anitbody, Rabbit Recombinant | 100 µg | 710487 |
Smooth Muscle Actin Monoclonal Anitbody, Rabbit Recombinant | 100 µg | 701457 |
MyQubit miRNA Assay
What it is
The MyQubit miRNA Assay for use with the Qubit® 2.0 Fluorometer allows easy and accurate quantitation of small amounts of miRNA even in the presence of ribosomal RNA, using a combination of existing Life Technologies reagents, common buffers, and the MyQubit miRNA Assay file. The MyQubit miRNA Assay is based on the Quant-iT™ OliGreen® ssDNA Reagent, which exhibits a large increase in fluorescence upon binding to nucleic acids.
What it offers
- Sensitivity—accurately detect as little as 0.5 ng miRNA, even in the presence of ribosomal RNA
- Simplicity—add your sample (in any volume between 1 μL and 20 μL), then read the concentration using the Qubit® 2.0 Fluorometer.
How it works
The MyQubit miRNA Assay is selective for small RNA, unlike other RNA detection methods such as the NanoDrop® UV-VIS spectrophotometer A260 assay or even the Qubit® RNA assays (see figure). The MyQubit miRNA Assay has a dynamic range of 5 to 500 ng/mL miRNA. The MyQubit miRNA Assay file can be downloaded from the bottom of the Qubit® 2.0 Fluorometer web page and permanently uploaded to your Qubit® 2.0 Fluorometer. In addition to the MyQubit miRNA Assay, the MyQubit firmware preloaded on all new Qubit® 2.0 instruments allows you to create other assays for your Qubit® 2.0 Fluorometer. Because the instrument is operated by simple commands, creating additional applications can be as straightforward as matching the spectral characteristics of the assay with the right LEDs and emission filters.
- Get instructions for downloading and installing the .qbt assay file from the MyQubit microRNA Assay manual (pdf)
- Download the MyQubit miRNA Assay .qbt file
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Comparison of detection techniques for accurate quantitation of small RNA in the presence of ribosomal RNA. rRNA at the concentrations listed was spiked into solutions containing 2 ng/µL siRNA, then read using the MyQubit miRNA assay, the Qubit® RNA assay, or by 260 nm absorbance (A260) on the NanoDrop® spectrophotometer. |
Product | Quantity | Cat. No. |
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Qubit® 2.0 Fluorometer | 1 instrument | Q32866 |
Quant-iT™ OliGreen® ssDNA Reagent |
1 mL | O7582 |
Silencer® Select GAPDH Positive Control siRNA (Hs, Mm, Rn) | each | 4390849 |
20X TE Buffer, RNase-free | 100 mL | T11493 |
ZOOM® CHAPS | 5 g | ZC10003 |
Qubit® Assay Tubes | 1 set | Q32856 |
New anti–human CD33 conjugates for flow cytometry
What it is
The Molecular Probes® portfolio of over 1,000 highly specific primary antibodies for flow cytometry is expanding to include more Research Use Only (RUO) selections. The anti–human CD33 monoclonal antibody (mouse-derived clone WM53) has been conjugated to a variety of fluorophores to expand your research options.
What it offers
- Trusted—Molecular Probes® brand
- Validated—all antibodies are tested in flow cytometry applications
- Selection—expanded offerings of primary antibody conjugates for flow cytometry
How it works
CD33 is a type I transmembrane glycoprotein and a member of the sialoadhesin family of cell surface receptors. It is absent from pluripotent stem cells but appears on myelomonocytic precursors after the appearance of CD34. It then continues to be expressed on both the myeloid and monocyte lineages, although it is absent from granulocytes.
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Product | Quantity | Cat. No. |
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CD33 Mouse Anti-Human mAb (clone WM53) PerCP-Cy®5.5 Conjugate | 0.1 mg | A15454 |
CD33 Mouse Anti-Human mAb (clone WM53) APC Conjugate | 100 tests | A15727 |
CD33 Mouse Anti-Human mAb (clone WM53) Fluorescein (FITC) Conjugate |
100 tests | A15763 |
CD33 Mouse Anti-Human mAb (clone WM53) PE Conjugate | 100 tests | A15795 |
CD33 Mouse Anti-Human mAb (clone WM53) PerCP Conjugate | 100 tests | A15804 |
Enzyme-mediated methodology for the site-specific radiolabeling of antibodies based on catalyst-free click chemistry
Zeglis BM, Davis CB, Aggeler R et al. (2013) Bioconjug Chem 24(6):1057–1067.
Antibodies are selective enough in their binding to offer the promise of an effective drug- and radioisotope-delivery system targeted at tumors. Researchers have long tried to exploit antibodies to do just that, and some have been successful. However, the lack of site selectivity when radiolabeling antibodies by most methods can result in inactivation of the antigen-binding domains, and this has slowed progress in the development of these potentially powerful tools.
Zeglis et al. have recently published a method for using catalyst-free click chemistry to radiolabel antibodies exclusively in the heavy-chain Fc domain, well separated from the antigen-binding domains. The method relies on the fact that IgG antibodies have a conserved N-linked glycosylation site on the CH2 domains of the heavy-chain Fc regions. The biantennary glycans that decorate those glycosylation sites are amenable to modification, and the method described is a stepwise enzymatic modification of the glycans, followed by a click chemistry process that results in radiolabels linked only to the glycosylation sites, well away from the antigen-binding sites.
We have taken this site-specific antibody labeling strategy and made it available in our SiteClick™ Antibody Labeling Kits. The SiteClick™ Antibody Labeling Kits make it easy for you to specifically label the Fc regions of IgG antibodies with R-phycoerythrin, any of an assortment of our most popular Alexa Fluor® dyes, or bright and photostable Qdot® nanocrystals.
- Watch a video about easy and site-specific labeling of an antibody using click chemistry
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Figure 1. The SiteClick™ antibody labeling system. The first step in the SiteClick™ antibody labeling process involves removal of terminal galactose residues from the heavy chain N-linked glycans using β-galactosidase, exposing essentially all possible modifiable GlcNAc residues. Second, the free terminal GlcNAc residues are activated with azide tags by enzymatic attachment of GalNAz to the terminal GlcNAc residues using the GalT(Y289L) enzyme. In the third step, the azide residues are reacted with the dibenzocyclooctyne (DIBO)-functionalized probe of choice (e.g., Alexa Fluor® 488 DIBO alkyne). The average degree of labeling is 3–3.5 labels per antibody. |
Select the right tools for oxidative stress detection
Generation of reactive oxygen species (ROS) is inevitable for aerobic organisms, but it occurs at a controlled rate in healthy cells. Under conditions of oxidative stress, ROS production is dramatically increased, resulting in alteration of membrane lipids, proteins, and nucleic acids. Oxidative damage is associated with aging as well as with a variety of pathological events, including atherosclerosis, carcinogenesis, ischemia reperfusion injury, and neurodegenerative disorders.
Reagent kits are optimized for flow cytometry or for fluorescence microscopy and microplate analysis.
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Imaging lipid peroxidation with the Image-iT® Lipid Peroxidation Kit. Human osteosarcoma (U2OS) cells were treated with 100 µM cumene hydroperoxide for 2 hr. A stain solution containing 10 µM Image-iT® Lipid Peroxidation Sensor and 2 drops/mL of NucBlue® Live ReadyProbes™ Reagent was applied for 30 min at 37°C. Cells were washed and imaged with Live Cell Imaging Solution. |
DEPARTMENTS
Model systems for Parkinson’s diseaseRecently, Life Technologies has partnered with the Parkinson’s Institute in Sunnyvale, California, to develop PD model systems using donor fibroblasts that have been collected at the Institute.
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Flow cytometric assays for apoptosisThe mechanisms and controls of programmed cell death—apoptosis—are under intense scrutiny, because defects in apoptosis appear to be involved in a wide variety of human disease processes.
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Reducing endogenous background with the Endogenous Biotin-Blocking Kit
A549 cells were fixed and permeabilized, treated (left) or not (right) with the Endogenous Biotin-Blocking Kit, labeled with a primary antibody against mouseanti-golgin-97 followed by DSB-X biotin–goat–anti-mouse IgG, then stained with Alexa Fluor ®488 streptavidin (green), Alexa Fluor® 647 phalloidin (magenta), andNucBlue® Fixed Cell Stain (blue). The image was acquired at 60x magnification on a Nikon® E800 upright microscope.
Highlight from BioProbes® Journal †
Live-cell staining of pluripotent stem cells: Alkaline Phosphatase Live Stain for early identification of induced pluripotent stem cells
In the Cell Signaling section of BioProbes 69, you will find the article “Live-cell staining of pluripotent stem cells". This article focuses on the Molecular Probes® Alkaline Phosphatase Live Stain, a fluorogenic alkaline phosphatase (AP) substrate that enables live-cell labeling of induced pluripotent stem cell (iPSC) colonies early in the reprogramming process, before immunocytochemical analysis is possible. When enzymatically turned over, this cell-permeant AP substrate produces a bright green-fluorescent product that then diffuses out of the cell, leaving behind no footprint.
AP activity has been shown to be up-regulated in pluripotent stem cells, including undifferentiated embryonic stem cells (ESCs), embryonic germ cells (EGCs), and iPSCs. Unlike other AP stains, neither the Alkaline Phosphatase Live Stain nor its enzymatic product accumulates in the cell. Alkaline Phosphatase Live Stain can be used to identify emerging iPSCs via robust fluorescent staining of the colonies as early as 14 days post-transduction, as well as to select iPSC colonies for expansion at 21 days post-transduction. Importantly, Alkaline Phosphatase Live Stain is nontoxic to emerging iPSCs and therefore can be used directly on master reprogramming plates to identify colonies for further propagation.
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Emerging iPSCs generated using the CytoTune®-iPS Sendai Reprogramming Kit. BJ human fibroblasts (ATCC) were transduced overnight using the CytoTune®-iPS Sendai Reprogramming Kit, and culture medium was replaced the next day. One week post-transduction, the cells were seeded onto inactivated MEF feeder cells in human PSC medium (DMEM/F-12 containing 20% KnockOut™ Serum Replacement and 4 ng/mL bFGF). At 14 days post-transduction, emerging iPSCs were analyzed for alkaline phosphatase activity using the Alkaline Phosphatase Live Stain, and images were collected on a Zeiss® Axiovert® microscope using a 10x objective: (A) phase-contrast image, (B) fluorescence image (using FITC optical filters), and (C) merged images.. |
† What's new with the BioProbes® Journal?
We are bringing our award-winning BioProbes® articles to you sooner. We will be publishing new BioProbes® articles online every month and highlighting those articles here. That way, we can keep you up-to-date on new fluorescence technologies and cell biology applications. Check back frequently and watch BioProbes 69 take shape!
Fluorophore selection in experimental design
Original broadcast: April 26, 2012
Choosing a fluorophore is one of the first important decisions to make when developing an experiment. A fluorophore is a compound that emits light at a specific wavelength when it has been excited at a lower wavelength. View the free webinar and explore:
- How to choose the best organic dye for an assay
- Quantum dots and how they compare to other dyes
- When to use a phycobiliprotein like R-PE or APC
- When to use a fluorescent protein like GFP
- How to choose a suitable dye to match your instrument
In addition, we explore the basic characteristics, strengths, and weaknesses of various fluorophores to help you develop the best assay for your needs.
An introduction to flow cytometric analysis using Molecular Probes® reagents, part I: Cell proliferation analysis
Original broadcast: November 21, 2011
In this free webinar, we discuss flow cytometric analysis of cell proliferation using CellTrace™ Violet and CellTrace™ CSFE, Click-iT® EdU, dual pulse labeling with EdU and BrdU, Vybrant® DyeCycle™ stains for live cell cycle analysis, and FxCycle™ stains for fixed cell cycle analysis.
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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* No purchase necessary. This promotion is only available to life science professionals 21 years or older in the US and Canada. Offer valid on requests received by Life Technologies no later than September 30, 2013, or until promotional supplies are depleted, whichever comes first. Limit 1 free copy per customer. Please allow 4–6 weeks for delivery. By accepting the Handbook, customers represent that they are not prohibited by law, regulation, or agency or institutional policy from receiving the Handbook from Life Technologies. Cannot be combined with other discounts or promotions. Offer void where prohibited, licensed, or restricted by federal, state, provincial, or local laws or regulation or agency/institutional policy. Other restrictions may apply.