In this issue

FEATURED NEW PRODUCTS

ReadyProbes™   Ready-to-use Alexa Fluor® dye–conjugated secondary antibodies—ReadyProbes™ fluorescent antibodies in convenient dropper bottles
     
ABfinity™ recombinant antibodies   ABfinity™ recombinant antibodies—New antibody for Bcl-2
     
MyQubit Amplex® Red Glucose/Glucose Oxidase Assay   Accurate glucose quantitation—MyQubit Amplex® Red Glucose/Glucose Oxidase Assay
     
detection of TCRαβ   Antibody conjugates for the detection of TCRαβ—New anti–mouse TCRαβ conjugates for flow cytometry

NEW APPLICATIONS

Mitophagy   Tools and techniques to measure mitophagy using fluorescence microscopy

PROVEN PERFORMERS

Tools fo apoptosis   A suite of tools for measuring apoptosis

UPCOMING EVENTS

flowcytometryUK 4th Meeting
July 29–31
Manchester, UK

OTHER PUBLICATIONS

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

FEATURED NEW PRODUCTS

Ready-to-use Alexa Fluor® dye–conjugated secondary antibodies—ReadyProbes™ fluorescent antibodies in convenient dropper bottles

What they are
Our Alexa Fluor® dye–conjugated secondary antibodies for immunocytochemistry are bright and often play an important role in published research. We took the brightest and most frequently used conjugates and put them into a ReadyProbes™ ready-to-use format. Supplied in dropper bottles, ReadyProbes™ reagents can eliminate the need to pipet solutions, make calculations, or prepare dilutions when you stain cells.


What they offer

  • Brilliant Alexa Fluor® secondary antibody conjugates, ready to use in dropper bottles
  • No dilutions, pipetting, or optimization required; just add, wash, and detect
  • Plenty of options for multiplexing

How they work
ReadyProbes™ Alexa Fluor® dye–conjugated antibodies are supplied ready to use, in dropper bottles. After incubation with the primary antibody is complete, simply add 2 drops of ReadyProbes™ antibody per milliliter of sample, incubate, wash, and image. For multiplexing, you can choose ReadyProbes™ formulations of either Alexa Fluor® 488 or 594 dye conjugated to anti–mouse IgG or anti–rabbit IgG antibodies that were raised in donkey or goat hosts.

 

ReadyProbes™ fluorescent antibodies  
Multiplex cell labeling using ReadyProbes™ fixation and staining reagents. Caki (human kidney carcinoma) cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit and then stained. Nuclei are stained with NucBlue® Fixed Cell ReadyProbes™ Reagent (blue), actin cytoskeleton is stained with ActinGreen™ 488 ReadyProbes™ Reagent (green), and mitochondria are labeled with mouse anti–ATP synthase subunit IF1 monoclonal antibody and detected with ReadyProbes™ Alexa Fluor® 594 goat anti–mouse IgG antibody (red).



ABfinity™ recombinant antibodies—New antibody for Bcl-2

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.

B-cell lymphoma 2 (Bcl-2) is the protein for which the Bcl-2 family of regulatory proteins is named. Primarily, Bcl-2 regulates apoptosis through the inhibition of cytochrome c release from mitochondria, via a feedback loop system involving caspases. Bcl-2 requires the BH4 motif for anti-apoptotic activity. During apoptosis, this motif is cleaved by caspases, causing the release of cytochrome c into the cytosol, which then further promotes caspase activity and, by extension, becomes pro-apoptotic. Bcl-2 also inhibits apoptosis by binding to the apoptotic protease–activating factor (APAF-1).


What they offer

  • Specificity—undergo rigorous validation
  • High performance—proven consistency from lot to lot
  • Efficiency—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 a mixture of recombinant monoclonal antibodies. This combines the improved signal strength that can come from using polyclonal antibodies, with the highly reproducible results you get from ABfinity™ monoclonal antibodies.

 

ABfinity™ recombinant antibody for Bcl-2  
Bcl-2 localization in HeLa cells. Immunocytochemistry analysis of HeLa cells probed with ABfinity™ Bcl-2 recombinant rabbit oligoclonal antibody using (A) Alexa Fluor® 488 goat anti–rabbit IgG as a secondary antibody (green), (B) DAPI to stain nuclei (blue), and (C) Alexa Fluor® 594 phalloidin to stain actin (red). (D) Composite image of the cells, showing cytoplasmic and nuclear localization of Bcl-2.



Accurate glucose quantitation—MyQubit Amplex® Red Glucose/Glucose Oxidase Assay

What it is
The MyQubit Amplex® Red Glucose/Glucose Oxidase Assay for use with the Qubit® 2.0 Fluorometer allows easy and accurate quantification of glucose or glucose oxidase using a combination of existing Life Technologies reagents, common buffers, and the MyQubit Amplex® Red Glucose/Glucose Oxidase Assay file. The MyQubit Amplex® Red Glucose/Glucose Oxidase Assay is based on the Amplex® Red Glucose/Glucose Oxidase Assay Kit, which uses an enzyme-coupled reaction to detect glucose or glucose oxidase. This assay is perfect for researchers who need to quantitate glucose concentrations from a few samples, whereas the Amplex® Red Glucose/Glucose Oxidase Assay Kit is perfect for researchers who have enough samples for a 96-well microplate.


What it offers

  • Sensitivity—accurate detection of glucose concentrations as low as 250 nM
  • Versatility—detection of glucose or glucose oxidase activity

How it works
The MyQubit Amplex® Red Glucose/Glucose Oxidase Assay has a measurement range of 250 nM to 50 mM glucose (see figure). The MyQubit Amplex® Red Glucose/Glucose Oxidase Assay file can be downloaded from the Qubit® 2.0 Fluorometer web page and permanently uploaded to your Qubit® 2.0 Fluorometer. In addition to the MyQubit Amplex® Red Glucose/Glucose Oxidase Assay, the MyQubit firmware that is 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.

 

MyQubit Amplex® Red Glucose/Glucose Oxidase Assay   The MyQubit Amplex® Red Glucose/Glucose Oxidase Assay. (A) The plot showing the line corresponding to the curve-fitting algorithm (a modified Hill plot) used to calculate concentration in the MyQubit Amplex® Red Glucose/Glucose Oxidase Assay. After using standards to create the curve, a dilution series of known concentrations was measured and plotted on the standard curve, demonstrating that the curve-fitting algorithm gives accurate values for quantitation. (B) The assay has the same look and feel as that of the existing Qubit® assays.



Antibody conjugates for the detection of TCRαβ—New anti–mouse TCRαβ conjugates for flow cytometry

What they are
We are expanding our Molecular Probes® portfolio of over 1,000 highly specific primary antibodies for flow cytometry to include more Research Use Only (RUO) selections. The anti–mouse TCRαβ antibody has been conjugated to a variety of fluorophores to expand your research options. The TCRαβ antibody, clone H57-597 (produced by Armenian hamster cells), has been shown to induce immature thymocytes to undergo apoptosis.


What they offer

  • Trusted—Molecular Probes® brand
  • Validated—all antibodies are tested in flow cytometry applications
  • Selection—expanded offerings of primary antibody conjugates for flow cytometry

How they work
Life Technologies primary antibodies provide direct detection of antigens. All Life Technologies antibodies designated “for flow cytometry” have been tested and validated for use in flow cytometry.

 

New Anti–Mouse TCRαβ Conjugates  


Flow cytometric analysis showing cells expressing the αβ heterodimer T cell receptor (TCRαβ). BALB/c splenocytes were double-stained with TCRαβ hamster anti-mouse mAb (clone H57-597), PE-Cy®5.5 conjugate and rat anti–mouse CD3–R-PE and analyzed by flow cytometry, gated on small lymphocytes.

 

NEW APPLICATIONS

Tools and techniques to measure mitophagy using fluorescence microscopy

Dolman NJ, Chambers KC, Mandavilli B et al. (2013) Autophagy 9(11). In press.

Mitophagy is a highly specialized and targeted form of macro-autophagy that degrades damaged or superfluous mitochondria. The correct functioning of mitophagy is essential for cell health, and deficits in this process may be responsible for human diseases such as Parkinson’s. Several key steps in mitochondrial clearance have been identified, including loss of mitochondrial membrane potential, stabilization of the kinase PINK1, and recruitment of parkin (PARK2). The E3 ligase activity of parkin causes recruitment of the autophagy receptor P62/SQSTM1 and subsequent binding to a key autophagy molecule, LC3B. These processes are easily studied using fluorescence microscopy. Recently Dolman et al. reviewed the myriad of fluorescent probes that can be used to study mitophagy, including probes to follow the recruitment of P62/SQSTM1 to damaged mitochondria and the subsequent trafficking to lysosomes (see figure below, A and B).

The figure shows HeLa cells treated with the protonophore CCCP to depolarize mitochondria. The mitochondria lose their characteristic “sausage-like” morphology and begin to form spheroids, many of which show colocalization of P62/SQSTM1 (B I–B IV, arrows and arrowheads). At this early time point, some (arrowhead) but not all (arrow) of these mitochondria are trafficked to lysosomes that are labeled with LysoTracker® Deep Red.

Mitophagy can also be studied using mitochondrial probes in combination with the autophagy marker LC3B (C). Here, cells transduced with BacMam CellLight® Mito-GFP and Premo™ Autophagy Sensor LC3B-RFP have been treated with CCCP. Mitochondrial spheroids are positive for LC3B-RFP, clear evidence for mitophagy.

 

Mitophagy

 

PROVEN PERFORMERS

A suite of tools for measuring apoptosis

Cell death cascades are complex and dynamic, underscoring the importance of a multiparametric approach for accurate assessment. Understanding the mechanisms of cell death and survival can represent a critical aspect of toxicological profiling and drug discovery.

Life Technologies offers a wide array of apoptosis assays for measuring multiple components on a choice of assay platforms. The breadth of apoptosis assays from Life Technologies offers you a range of technologies to give you the right solution for your research.

Apoptosis measurement tools:

  • Caspase activity assays—measure a distinctive feature of early apoptosis
  • Annexin V staining—identify an intermediate apoptotic stage
  • DNA fragmentation and morphology probes—characterize changes in late apoptosis
  • Mitochondrial function assays—measure changes that usually occur early in apoptosis

Use the selection guides for apoptosis products to compare product features and review citations from other users.

CellEvent® Caspase-3/7 Green Detection Reagent


Multiplex staining of apoptotic cells. CellEvent® Caspase-3/7 Green Detection Reagent can be used in a simple no-wash protocol to monitor apoptosis in real time using fluorescence microscopy, flow cytometry, or microplate analysis.

 

DEPARTMENTS

On the web

Cytotoxicity assay guides

 

Cytotoxicity assay selection guides

Cytotoxicity assays are widely used in fundamental research and in drug discovery to screen libraries for toxic compounds. Reagents and assays from Life Technologies can be used to evaluate many aspects of cytotoxicity, broadly grouped on our cytotoxicity page into 5 application categories: viability, proliferation, mitotoxicity, lipotoxicity, and genotoxicity. Within each of those categories are handy selection guides to cytotoxicity measures to help you find the right viability or cytotoxicity assays for your research.

Imaging corner

Immunocytochemical analysis using anti–Rb [pT821] ABfinity™ antibodies
Click to enlarge
 

Immunocytochemical analysis using anti–Rb [pT821] ABfinity™ antibody

U2OS cells were labeled with either (A) anti–Rb [pT821] ABfinity™ recombinant rabbit oligoclonal antibody or (B) anti–Rb [pT821] plus a competing Rb phosphopeptide. Following the primary antibody incubation, both samples were labeled with Alexa Fluor® 488 goat anti–rabbit IgG secondary antibody (green), as well as with the actin stain Alexa Fluor® 594 phalloidin (red) and DAPI nuclear stain (blue).

The composite images show nuclear localization of Rb [pT821]; the lack of antibody signal in cells treated with both antibody and phosphopeptide demonstrates the antibody’s specificity. Similar results are seen with the anti–Rb [pT821] ABfinity™ recombinant rabbit monoclonal antibody.

Highlight from BioProbes® Journal

ReadyProbes™ ready-to-use imaging reagents for brilliant results: Simply add and image

In the Cell Imaging section of BioProbes 69, the article “ReadyProbes™ ready-to-use imaging reagents for brilliant results” describes a family of products designed to simplify your fluorescence imaging workflow. These ReadyProbes™ products include NucBlue®, NucGreen™, and NucRed™ nuclear stains for the blue, green, and red fluorescence channels, respectively, ActinGreen™ and ActinRed™ reagents for bright labeling of F-actin, and CellEvent® Caspase-3/7 Green reagent for tracking the early stages of apoptosis. These are in addition to the ReadyProbes™ Alexa Fluor® dye–conjugated secondary antibodies described in the Featured New Products section of this ProbesOnline™ newsletter.

ReadyProbes™ fluorescent labeling reagents are provided as user-friendly formulations in convenient dropper bottles, allowing you to stain cells without pipetting solutions, making calculations, or preparing dilutions. Simply apply 2 drops per milliliter of sample, and you're ready for imaging. Whether you are new to fluorescence imaging or an experienced user trying to streamline your protocols, ReadyProbes™ reagents allow you to spend less time preparing solutions and more time analyzing results.

ReadyProbes™ ready-to-use imaging reagents   Three-color imaging of actin (green), nuclei (blue), and peroxisomes (red) in fixed BPAE cells. BPAE cells were fixed, permeabilized, and blocked using the Image-iT® Fixation/Permeabilization Kit. Peroxisomes were labeled using a rabbit anti-PMP70 antibody followed by detection with Alexa Fluor® 594 goat anti–rabbit IgG secondary antibody (ReadyProbes™ formulation). Actin was stained using ActinGreen™ 488 ReadyProbes™ Reagent, and nuclei were counterstained with NucBlue® Fixed Cell ReadyProbes™ Reagent.

† 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!

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