ProbesOnline™ August 2013—Onstage incubator for time-lapse imaging, chemiluminescence ELISAs, oxidative stress detection

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

The EVOS® FL Auto Imaging System with the EVOS® Onstage Incubator.

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

DynaLight™ glow and flash protocols. DynaLight™ Substrate with RapidGlow™ Enhancer can be used either in glow or flash mode. In glow mode, maximum signal is achieved after 2 minutes at 37°C or 5–10 minutes at room temperature. The flash signal is generated on-demand with the addition of DynaLight™ Trigger Solution as early as 5 seconds after DynaLight™ Substrate addition.

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.

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.

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

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.

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.

Staining of normal human peripheral blood cells with staining buffer (autofluorescence, open green histogram) or anti–human CD33 (clone WM53) PerCP-Cy®5.5 conjugate (filled blue histogram). Cells in the monocyte gate were used for analysis.

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

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.

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.

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.

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.