In This Issue

FEATURED NEW PRODUCTS

EVOS® FL Auto Cell Imaging System EVOS® FL Auto Cell Imaging System—the power of automation with the simplicity of EVOS® technology
   
SiteClick™ Antibody Labeling Kits Site-specific antibody labeling with Qdot® reagents—SiteClick™ Antibody Labeling Kits
   
Pacific Green™ for flow cytometry New violet laser–excitable dye to expand your multicolor capacity—Pacific Green™ dye and conjugates for flow cytometry
   
New antibodies for Cyclin D1 ABfinity™ recombinant antibodies—new antibodies for cyclin D1
   
New Antibodies See all of this month's new antibodies

 

NEW APPLICATIONS

pHrodo™ Red pH indicatorProperties of ribbon and non-ribbon release from rod photoreceptors revealed by visualizing individual synaptic vesicles

 

PROVEN PERFORMERS

Ion indicators Choose ion indicators for your experiments using new web-based product guides

 UPCOMING MEETINGS

International Society for Advancement of Cytometry (ISAC/CYTO)
May 19–23  |   San Diego, CA
Booth #624

American Society for Microbiology 112th General Meeting (ASM)
May 18–21   |   Denver, CO
Booth #736

International Society for Stem Cell Research (ISSCR)
June 12–15   |   Boston, MA
Booth #630

OTHER PUBLICATIONS

BioProbes® Journal of Cell Biology Applications
BioProbes 69  
 
The Molecular Probes® Handbook
Molecular Probes Handbook  

FEATURED NEW PRODUCTS

EVOS® FL Auto Cell Imaging System—the power of automation with the simplicity of EVOS® technology

What it is
Eliminate the complexities of microscopy without compromising performance. Regardless of your application or budget, there’s an EVOS® cell imaging instrument that brings simplicity right to your bench.



What it offers

  • Powerful features—automated system capable of time-lapse imaging, plate scanning, automated cell counting, tile stitching, and Z-stacks
  • Affordability—excellent value makes automated cell imaging accessible to labs of all sizes and budgets
  • Simplicity—intuitive user interface allows novices or experts to generate high-quality single and time-lapse images in a snap


How it works

By reducing the complexities of fluorescence microscopy, the affordable, intuitive EVOS® Cell Imaging Systems have revolutionized the way researchers use fluorescence microscopy to image cells. The latest addition to the EVOS® line, the EVOS® FL Auto Cell Imaging System brings the simplicity of EVOS® technology to an automated imaging platform capable of plate scanning, automated cell counting, image stitching, and time-lapse imaging—with a simple touch of the monitor. Containing both color and monochrome cameras, the EVOS® FL Auto Cell Imaging System is the perfect instrument for both fluorescent and colorimetric/transmitted-light imaging.

 

EVOS® FL Auto Cell Imaging System 
The intuitive user interface on the EVOS® FL Auto Cell Imaging System allows users to quickly and easily capture images.



Site-specific antibody labeling with Qdot® reagents—SiteClick™ Antibody Labeling Kits

What they are
SiteClick™ Antibody Labeling Kits allow you to conjugate your IgG antibodies to the bright and photostable red and near-IR fluorescent Qdot® reagents.



What they offer

  • Simplicity—in 3 simple steps, expertly attach Qdot® particles or R-PE to your antibody
  • Quality conjugates—enzymatic labeling system keeps antibodies and antigen-recognition site intact
  • Superior brightness and photostability—when excited at the excitation maxima and detected with optimized Qdot® filters, Qdot® reagents offer unparalleled brightness and photostability compared to standard fluorophores such as FITC or even Alexa Fluor® 488


How they work

In the first step of SiteClick™ conjugation, the Fc region of the antibody is modified by β-galactosidase.
The azide-containing sugar, UDP-GalNAz, is then added to the modified carbohydrate domain of the antibody via the β-1,4-galactosyltransferase (Gal-T)-catalyzed reaction targeting the specific N-linked oligosaccharide GlcNAc residues. This specific targeting maintains the integrity of the antigen-binding site on the antibody. Finally, the antibody (now containing an azide moiety) is conjugated to the DIBO-modified label (Qdot® reagent) in a copper-less click reaction with simple overnight incubation.

 

SiteClick™ Antibody Labeling Kits 
HeLa cells labeled with Qdot® 655 using the SiteClick™ kit. A mouse primary antibody, anti–golgin-97, was labeled using the SiteClick™ Qdot® 655 Antibody Labeling Kit, and the resulting conjugate was incubated with fixed and permeabilized HeLa cells at a final concentration of 10 nM (magenta fluorescence). Cells were counterstained with NucBlue® Live  ReadyProbes™ Reagent (blue fluorescence) and ActinGreen™ 488 ReadyProbes™ Reagent (green fluorescence).



New violet laser–excitable dye to expand your multicolor capacity—Pacific Green™ dye and conjugates for flow cytometry

What it is
The Molecular Probes® dye portfolio now supports immunophenotyping by flow cytometry with three dyes for the violet laser. Pacific Green™ dye joins Pacific Orange™ and Pacific Blue™ dyes, which are established performers optimally excited by the 405 nm laser.



What it offers

  • Increased flexibility in multicolor experiments
  • Detection in the second emission channel (ex/em: 411 nm/510 nm)
  • Compatibility with Pacific Blue™ dye and Pacific Orange™ dye
  • Optimal for use on any flow cytometer equipped with a violet laser


How it works

Pacific Green™ dye is excited at 411 nm and emits at 500 nm. Pacific Blue™, Pacific Green™, and Pacific Orange™ dye conjugates can be simultaneously excited at 405 nm for emission at 455 nm, 500 nm, and 551 nm, respectively, facilitating three-color analysis.

Multiple product formats are available to fit most applications: primary conjugates (Hu and Ms), secondary conjugates (goat anti-mouse and streptavidin), and labeling reagents (succinimidyl ester and Zenon® mouse IgG1 labeling kit).

 

Pacific Green dye conjugates 
Excitation spectra (dotted lines) and emission spectra (solid lines) for Molecular Probes® violet laser–excited Pacific Blue™ dye (blue), Pacific Green™ dye (green), and Pacific Orange™ dye (orange). The shaded area represents the emission bandpass filter (522/31) used for detection of the Pacific Green™ dye.



ABfinity™ recombinant antibodies—new antibodies for cyclin D1

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. Over the past year we have launched several new ABfinity™ recombinant antibodies.

Cyclin D1 is a member of the cyclin family and functions as the regulatory component of CDK kinase. The cyclin D1-CDK4 complex phosphorylates proteins of the Rb family to regulate the G1/S transition of the cell cycle. In addition, cyclin D1 positively regulates protein phosphorylation, mammary gland epithelial cell proliferation, and fat cell differentiation. In humans, the CCND1 gene encoding cyclin D1 is located on chromosome 11.



What they offer

  • Specificity—undergo rigorous validation
  • High performance—proven consistency from lot to lot
  • Efficiency—detect low-level targets, with less sample


How they work

ABfinity™ antibodies are manufactured by transfecting mammalian cells with high-level expression vectors containing immunogen-specific heavy- and light-chain antibody cDNA. This production process offers consistent lot-to-lot antibody performance.

ABfinity™ oligoclonal antibodies are a mixture of recombinant monoclonal antibodies. This combines the improved signal strength that can come from using a polyclonal with the highly reproducible results you get from ABfinity™ monoclonal antibodies.

 

Cyclin D1 ABfinity™ Recombinant Rabbit Monoclonal Antibody 
Immunocytochemistry analysis of HeLa cells stained with Cyclin D1 ABfinity™ Recombinant Rabbit Monoclonal Antibody. Alexa Fluor® 488 goat anti-rabbit was used as the secondary antibody (green) (A), DAPI was used to stain the nucleus (blue) (B), and Alexa Fluor® 594 phalloidin was used to stain actin (red) (C). (D) Composite image of cells showing nuclear localization of cyclin D1.

 

NEW APPLICATIONS

Properties of ribbon and non-ribbon release from rod photoreceptors revealed by visualizing individual synaptic vesicles

Chen M, Van Hook MJ, Zenisek D, Thoreson WB

The release of synaptic vesicles by rod photoreceptors is mediated by Ca2+ flux through L-type channels, which is triggered by membrane potential changes upon illumination. In rod photoreceptors, this release appears to be associated with synaptic ribbons—electron-dense structures that function in the delivery, priming, and clustering of vesicles near the L-type Ca2+ channels—as well as with non-ribbon sites. Using total internal reflection fluorescence microscopy (TIRFM), Chen and coworkers characterized the kinetics of vesicle release from ribbon and non-ribbon sites in salamander rods by visualizing individual synaptic vesicles loaded with either pHrodo™ Red 10,000 MW dextran or the FM® 1-43 membrane probe. Their vesicle-loading protocol included short incubation times (3 minutes for pHrodo™ dextran, 1 minute for FM® 1-43) in order to label only a fraction of the ~100,000 vesicles in the rod terminal. With only a small number of vesicles labeled, they were able to visualize individual vesicles as they approached, paused, and then fused with the plasma membrane. Vesicle release was also studied with longer loading times that labeled a larger population of vesicles (Figure 1).

The FM® 1-43 membrane probe is well established for the study of actively firing neurons. This nonfluorescent, water-soluble dye inserts into the outer leaflet of the cell membrane, where it becomes intensely fluorescent. During neuronal endocytosis, FM® 1-43 becomes internalized in the recycled synaptic vesicle membranes, producing bright staining of the nerve terminals (typically seen as green fluorescence, though it depends on the optical filters used). The 10,000 MW dextran conjugated to the pHrodo™ Red pH indicator (a pH-sensitive rhodamine derivative) is a new endocytosis marker that exhibits bright red fluorescence once it is incorporated into acidic organelles such as endosomes and synaptic vesicles but is only weakly fluorescent in extracellular medium (pH 7.8).

The authors validated the use of pHrodo™ Red dextran for visualizing synaptic vesicles by showing that, when used simultaneously, the pHrodo™ Red dextran and FM® 1-43 probes fluorescently labeled the same set of organelles. They also report that vesicles labeled with pHrodo™ Red dextran were slightly less fluorescent that those labeled with FM® 1-43 but provided a better signal-to-noise ratio, possibly due to residual FM® 1-43 membrane staining and more cellular autofluorescence at the FM® 1-43 wavelengths. After compensating for the lower pHrodo™ signals with slightly longer frame durations of 40 msec, they chose to conduct the majority of their vesicle-labeling experiments with pHrodo™ Red dextran.

 

pHrodo™ Red 10,000 MW dextran
Vesicle release sites in rod photoreceptor terminals revealed with pHrodo™ Red 10,000 MW dextran. Synaptic vesicles of salamander rods were loaded with pHrodo™ Red 10,000 MW dextran (500 μg/mL) by incubation at room temperature for 30 min in darkness. Intact rod photoreceptors are depolarized in darkness, promoting synaptic vesicle exocytosis and endocytosis. After loading, rods were placed in a low-Ca2+ solution, exposed to light to inhibit further synaptic release, and then enzymatically isolated and plated on high NA glass coverslips. pHrodo™ Red dextran–loaded vesicles were illuminated with a 561 nm solid-state laser (Melles Griot) and then visualized by TIRFM using a 1.65 NA objective (Olympus). The footprint of the rod terminal attached to the cover glass is outlined in white. Release was stimulated by a 1 sec application of 50 mM KCl. Upon depolarization, vesicles advanced toward the plasma membrane, causing a fluorescence increase. After a brief pause at the membrane surface, vesicles then fused, causing a decrease in fluorescence (not shown). In this example, vesicle release sites were mapped by measuring the depolarization-evoked increase in near-membrane fluorescence. Release occurred mostly near the center of the terminal (middle arrow) but was also observed in other parts of the terminal (left and right arrows). Scale bar: 1 μm. Figure provided by Minghui Chen and Wallace B. Thoreson, University of Nebraska Medical Center, Omaha, Nebraska, and reproduced with permission.

PROVEN PERFORMERS

Choose ion indicators for your experiments using new web-based product guides

Determining changes in ionic concentrations with both spatial and temporal resolution has become critical in many research areas, from drug discovery to neuronal functional studies. To help you choose the correct ion indicator for your experiment, we have developed a series of uncomplicated selection guides for calcium, sodium, magnesium, zinc, and pH indicators. These guides list the important properties of each indicator and provide key usage notes. You’ll be able to see excitation/emission wavelengths, whether an indicator is cell permeant or impermeant, what the readout is (increase in emission or shift in excitation), and the appropriate concentration range for detection.

 

Oregon Green® 488 BAPTA-1 
Confocal line scan image of calcium “puffs” in a Xenopus oocyte, using Oregon Green® 488 BAPTA-1.

 

DEPARTMENTS

On the Web

CellEvent® Caspase-3/7 Reagent

 

See how researchers are using a fluorescent caspase detection reagent to monitor apoptosis

Since its release, CellEvent® Caspase-3/7 Green Detection Reagent has been used by researchers like you as a simple, effective method for monitoring apoptosis in live or fixed cell samples. View the growing citation list to see how researchers have used CellEvent® Caspase-3/7 Green Detection Reagent in their experimental systems.

Imaging Corner

p62-positive protein aggregate accumulation during chloroquine-mediated autophagy block
Click to enlarge
 

p62-positive protein aggregate accumulation during chloroquine-mediated autophagy block

A549 cells were transduced with Premo™ Autophagy Sensor GFP-p62 (green) and CellLight® Tubulin-RFP (red) and cultured for 24 hr. Chloroquine was added to a final concentration of 60 µM, and cells were cultured for a further 16 hr before counterstaining with Hoechst® 33342 (blue). p62-positive protein aggregates can be seen associated with microtubules.

Highlight From BioProbes® Journal*

Universal site-selective labeling of any antibody: A new paradigm

In the Protein Expression section of BioProbes 69, we introduce the new SiteClick™ antibody labeling system in the article “Universal site-selective labeling of any antibody”. This modular, click chemistry–mediated method allows you to enzymatically label essentially any antibody on its heavy chain N-linked glycans. In contrast to standard antibody labeling techniques—which can be cumbersome, inconsistent, and detrimental to antigen-binding domains—the SiteClick™ site-selective approach produces highly robust and reproducible labeling of antibodies with an impressive choice of detection molecules, including R-phycoerythrin (R-PE), Qdot® probes, fluorescent dyes, and biotin.

Our newest SiteClick™ products include R-PE antibody labeling kits, which are designed especially for flow cytometry applications, as well as several Qdot® antibody labeling kits that produce conjugates for imaging or flow cytometry applications. These kits provide the key reagents for site-selective labeling of 100 μg of purified antibody with either R-PE or Qdot® fluorophores. In practice, we find the degree of labeling of mouse and rabbit monoclonal antibodies to be consistently between 3 and 4 labels per antibody. The kit’s easy-to-follow workflow is compatible with antibodies from a number of different species as well as with several antibody classes, allowing novice and experienced scientists alike to obtain efficient antibody labeling every time.


SiteClick™ Antibody Labeling Kits 

 

Immunocytochemistry with SiteClick™ labeled antibodies. HeLa cells were fixed, permeabilized, and incubated with 10 nM Qdot® 655 anti–golgin-97 antibody (magenta). This conjugate was generated using the SiteClick™ Qdot® 655 Antibody Labeling Kit and mouse monoclonal anti–golgin-97 antibody (clone CDF3). After antibody incubation, the cells were counterstained with NucBlue® Live (blue) and ActinGreen™ 488 (green) ReadyProbes™ reagents prior to imaging.

  

* What's new with the BioProbes® Journal?

We are bringing our award-winning BioProbes® articles to you sooner. Instead of producing two print editions per year, 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!

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*This promotion is available only to life science professionals 21 years or older in the US (excluding Puerto Rico) and Canada who submit a completed request form. Limit 1 copy per customer.  Offer void where prohibited, licensed or restricted by federal, state, provincial, or local laws or regulation or agency/institutional policy. Other restrictions may apply. Offer is valid until June 30, 2013, or while Handbook supplies last, whichever comes first.