In this issue

FEATURED NEW PRODUCTS

  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
     
  ABfinity™ recombinant antibodies—New antibodies for smooth muscle actin
     
  Accurate microRNA quantitation—MyQubit miRNA Assay
     
  Antibody conjugates for the detection of CD33—New anti–human CD33 conjugates for flow cytometry

NEW APPLICATIONS

Enzyme-mediated methodology for the site-specific radiolabeling of antibodies based on catalyst-free click chemistry

PROVEN PERFORMERS

  Select the right tools for oxidative stress detection

 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

The Molecular Probes® Handbook

FEATURED NEW PRODUCTS

Keep your cells healthy during imaging, and see the results in time lapse

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.


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

FLoid® Cell Imaging Station rides the BioBus to inspire love of science

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.



Developing sensitive chemiluminescence ELISAs

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.

 


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.



ABfinity™ recombinant antibodies

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.



Accurate microRNA quantitation

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.

 
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.



Antibody conjugates for the detection of CD33

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.

NEW APPLICATIONS

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.

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.

PROVEN PERFORMERS

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.

 
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

On the web

 

Model systems for Parkinson’s disease

Recently, 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.


 

Flow cytometric assays for apoptosis

The 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.

Imaging corner

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 mouse anti-golgin-97 followed by DSB-X biotin–goat–anti-mouse IgG, then stained with Alexa Fluor ®488 streptavidin (green), Alexa Fluor® 647 phalloidin  (magenta), and NucBlue® 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.

   
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!

Molecular Probes® webinar series

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.