In This Issue

FEATURED NEW PRODUCTS

Image-iT® Lipid Peroxidation Kit Ratiometric Quantitation of Lipid Peroxidation in Live Cells—Image-iT® Lipid Peroxidation Kit
   
pHrodo™ Red and Green Reactive Dyes Visualize Phagocytosis, Endocytosis, and Internalization—pHrodo™ Red and Green Reactive Dyes
   
Alexa Fluor® 680 and 790 Antibody Labeling Kits Simple, Reliable IgG Labeling With Near-IR Dyes—Alexa Fluor® 680 and 790 Antibody Labeling Kits
   
ABfinity™ Recombinant Phosphospecific Antibodies Detect EIF2a Phosphorylation—New ABfinity™ Recombinant Monoclonal Antibodies
   
PerCP-Cy®5.5 Conjugates for Flow Cytometry Bright Conjugates for 21 Key CD Markers and Cytokines—New PerCP-Cy®5.5 Conjugates for Flow Cytometry
   
New Antibodies See all of this month's New Antibodies

 

NEW APPLICATIONS

Tali® Image-Based Cytometer Firmware Update New Cell Cycle Analysis Capability—Tali® Image-Based Cytometer Firmware Update

 

PROVEN PERFORMER

Luminex® Assays Using Molecular Probes® SAPEAchieve More Consistent Luminex® Assays Using Molecular Probes® SAPE

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OTHER PUBLICATIONS

BioProbes® Journal for Cell Biology Applications
BioProbes 67 
 
The Molecular Probes® Handbook
Molecular Probes Handbook
 

 

FEATURED NEW PRODUCTS

Ratiometric Quantitation of Lipid Peroxidation in Live Cells—Image-iT® Lipid Peroxidation Kit

What It Is
The Image-iT® Lipid Peroxidation Kit enables the accurate detection of lipid peroxidation in live cells. This reagent localizes to membranes of live cells and undergoes a green to red fluorescence shift when oxidized.



What It Offers

  • Accurate—ratiometric detection of lipid peroxidation in live cells
  • Easy to use—simply add reagent to the complete medium and measure lipid peroxidation in 30 minutes
  • Ready to use—available as a 1,000X stock solution in DMSO; cumene hydroperoxide is provided as a positive control


How It Works

The Image-iT® Lipid Peroxidation Kit supplies BODIPY® 581/591 C11 reagent, which localizes to membranes of live cells. Upon oxidation by lipid hydroperoxides, the reagent undergoes a shift in fluorescence from red (~590 nm) to green (~501 nm), providing a ratiometric indication of lipid peroxidation that is compatible with traditional and high-content microscopy as well as flow cytometry.

 

Image-iT® Lipid Peroxidation Kit Lipid peroxidation detection with the Image-iT® Lipid Peroxidation Kit. Human osteosarcoma (U2OS) cells were plated on 35 mm glass-bottom dishes (MatTek) and stained with 10 µM Image-iT® Lipid Peroxidation Sensor for 30 min in complete growth medium at 37°C. The cells were then treated with (A) vehicle (ethanol) or (B) 200 µM cumene hydroperoxide for 2 hr at 37°C. Hoechst® 33342 was added for the last 30 min of the incubation period. Cells were washed three times with phosphate-buffered saline (PBS) and imaged on a Zeiss® Axiovert® inverted microscope using a 40x objective and filters for Hoechst®, FITC, and Texas Red® dye channels.



Visualize Phagocytosis, Endocytosis, and Internalization—pHrodo™ Red and Green Reactive Dyes

What They Are
Our pHrodo™ STP esters and maleimides are reactive versions of our unique fluorogenic pH indicators developed to allow researchers to label protein or any amine- or thiol-containing biomolecule. The addition of pHrodo™ green indicators expands the multiplexing capabilities of this technology for the study of many membrane internalization processes.



What They Offer

  • Flexibility—label any amine- or thiol-containing molecule with a pH indicator dye
  • Multiplexing capability—available in red- and green-fluorescent forms
  • Utility—ideal for studying membrane transfer and internalization processes


How They Work

The pHrodo® green and red maleimides and amine-reactive esters can be used in a simple protocol for labeling reactions. The dyes are provided as dry powders that are reconstituted in DMSO to make the stock solutions. Protein to be labeled with the amine-reactive forms of pHrodo™ dye should be in 0.1 M sodium bicarbonate buffer, pH 8.3, to a concentration of ≥1 mg/mL. The reactive dye is added to the protein solution and reacted for up to an hour. The resulting conjugates can be purified using standard gel filtration techniques. Thiols can be labeled in a similar manner; however, a protein reduction step may be required prior to the labeling reaction (see product manual).

 

pHrodo™ dye–based detection of phagocytosis 
Schematic of pHrodo™ dye–based detection of phagocytosis. Particles (microorganisms or proteins) labeled with pHrodo™ dye are added to cells. Some labeled particles remain in solution or become nonspecifically attached to cells; they do not fluoresce because of the neutral pH of the extracellular environment. Other labeled particles are taken up by phagocytosis and become encapsulated in vesicles. As vesicles are processed, pH decreases and the pHrodo™ dye–labeled particles fluoresce bright red (pHrodo red dye) or bright green (pHrodo green dye).



Simple, Reliable IgG Labeling With Near-IR Dyes—Alexa Fluor® 680 and 790 Antibody Labeling Kits

What They Are
Alexa Fluor® 680 and 790 Antibody Labeling Kits allow you to label any IgG with near-infrared fluorescent dyes. The emission of Alexa Fluor® 680 and 790 dyes makes antibodies labeled with these fluorophores useful for most small animal in vivo imaging applications and platforms and also for multicolor fluorescence western detection using systems such as the LI-COR® Odyssey® infrared imaging system.



What They Offer

  • Rapid labeling—a simple protocol gives you labeled antibodies, typically in ~90 min, with only 15 min of hands-on time
  • Stable fluorescent conjugates—reliable reagents for many fluorescence-based imaging and flow cytometry applications
  • Complete kits—all labeling components are provided, including dye, purification resin, and tubes


How They Work

Molecular Probes® Alexa Fluor® Antibody Labeling Kits contain everything you need to perform five separate labeling reactions and to purify the resulting conjugates. Each reaction is optimized for labeling 100 μg of antibody (or other molecules >40 kDa). The resulting conjugates are ideal for multiple applications, including flow cytometry, fluorescence microscopy, immunohisto/immunocytochemistry, primary detection, ELISA, indirect FISH, small animal in vivo imaging, and fluorescent western blot detection.

Alexa Fluor® 680 and 790 Antibody Labeling Kits  
Fluorescence emission spectra of (A) Alexa Fluor® 680 and (B) Alexa Fluor® 790 goat anti–mouse IgG antibodies in pH 7.2 buffer.
Product* Quantity Cat. No.
Alexa Fluor® 680 Antibody Labeling Kit
5 labeling reactionsA20188
Alexa Fluor® 790 Antibody Labeling Kit
5 labeling reactionsA20189
*Note: In addition to the Alexa Fluor® 680 and 790 Antibody Labeling Kits, we offer ten other Alexa Fluor® dye, Pacific Blue™ dye, and Pacific Orange™ dye Antibody Labeling Kits, developed for efficient labeling of ~100 µg of protein. These kits were formerly called Monoclonal Antibody Labeling Kits; all have now been renamed Antibody Labeling Kits. See all Antibody Labeling Kits.



Detect EIF2a Phosphorylation—New ABfinity™ Recombinant Monoclonal Antibodies

What They Are
EIF2a (eukaryotic initiation factor 2A) is a heterotrimer composed of three subunits (alpha, beta, and gamma). This translation initiation factor was purified from rabbit reticulocytes and has been shown to drive binding of initiator methionyl-tRNA (met-tRNAi) to the 40S ribosome in an AUG-dependent manner to form the 43S pre-initiation complex. The alpha subunit of EIF2a acts as the regulatory subunit and can be phosphorylated by related protein kinases upon activation in response to stress. Phosphorylated EIF2a inhibits EIF2b activity and prevents guanine nucleotide exchange. Our EIF2a ABfinity™ recombinant rabbit antibodies offer researchers a way to detect EIF2a phosphorylation.



What They Offer

  • Reliability—reproducible lot-to-lot performance
  • Consistency—minimize the need to revalidate working antibody dilutions for your experiments each time you order


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. ABfinity™ oligoclonal antibodies are a mixture of recombinant monoclonal antibodies, combining the improved signal strength of a polyclonal antibody with the highly reproducible results you get from ABfinity™ monoclonal antibodies.

 

ABfinity™ Recombinant Rabbit Monoclonal Antibody 
Immunocytochemistry analysis with EIF2a [pS51] ABfinity™ Recombinant Rabbit Monoclonal Antibody. U2OS cells were labeled with EIF2a [pS51] ABfinity™ Recombinant Rabbit Monoclonal Antibody and visualized with (A) Alexa Fluor® 488 goat anti-rabbit (green). (B) DAPI was used to stain the nucleus (blue). (C) Alexa Fluor® 594 phalloidin was used to stain actin (red). Panel D is the composite image, showing nuclear localization of EIF2a.



Bright Conjugates for 21 Key CD Markers and Cytokines—New PerCP-Cy®5.5 Conjugates for Flow Cytometry

What They Are
Antibodies to key CD markers and cytokines are now available conjugated to the PerCP-Cy®5.5 fluorophore.



What They Offer

  • Brightness—increased brightness compared to comparable PE-Cy®5.5 conjugates
  • Convenience—packaged in 25 µg sizes


How They Work

PerCP-Cy®5.5 is a tandem conjugate that combines the peridinin chlorophyll protein (PerCP) complexes found in nature with the Cy®5.5 cyanine dye, thus permitting simultaneous multicolor labeling and detection of multiple targets with excitation by a single excitation source—the 488 nm line of the argon-ion laser.

  

PerCP-Cy®5.5 Conjugates for Flow Cytometry 
Flow cytometry analysis of cells stained with PerCP-Cy®5.5 conjugate. Normal human peripheral blood cells were left unstained (outlined histogram, to show autofluorescence) or stained with mouse anti–human CD45 PerCP-Cy®5.5 (filled histogram). Cells were gated on lymphocytes and subjected to further analysis.
Product Quantity Cat. No.
CD11b Rat Anti-Mouse mAb (clone M1/70), PerCP-Cy®5.5 Conjugate25 testsA14787
CD11c Hamster Anti-Mouse mAb (clone N418), PerCP-Cy®5.5 Conjugate25 µgA14788
CD127 (IL-7Rα) Rat Anti-Mouse mAb (clone A7R34), PerCP-Cy®5.5 Conjugate25 µgA14799

NEW APPLICATIONS

New Cell Cycle Analysis Capability—Tali® Image-Based Cytometer Firmware Update 2.0 is Available

The Tali® Image-Based Cytometer is a three-channel quantitative cell analysis instrument designed to perform cell counting, fluorescent protein (GFP and RFP) detection, viability assays, and apoptosis determination. With the 2.0 firmware upgrade, you can perform cell cycle analysis, fine-tune the fluorescence thresholds, and adjust the x-axis.

The Tali® instrument can now quantify the number of propidium iodide (PI)-stained cells in all phases of the cell cycle. These cell cycle data are in FCS format and can be analyzed using any flow cytometry modeling software. The ability to adjust the threshold, along with the visual confirmation the instrument provides, results in reproducible selection of the correct fluorescence threshold. Changing the x-axis allows you to zoom in on regions of interest.

 

Tali® Image-Based Cytometer Firmware Update 
New cell cycle analysis capabilities of the Tali® Image-Based Cytometer. Results of a cell cycle experiment using propidium iodide–stained cells are shown. Users can launch a pop-up key pad that allows adjustment of the fluorescent thresholds and x-axis. The color-coded areas of the histogram correspond to different cell cycle phases.

PROVEN PERFORMER

Achieve More Consistent Luminex® Assays Using Molecular Probes® SAPE

Accurate, reliable data from Luminex® multiplex assays depend on consistent detection reagents. Since 1995, Molecular Probes® Streptavidin R-PE (SAPE) has been manufactured using a rigorous conjugation and dye chemistry methodology and undergoes extensive purification, resulting in a consistent, high-quality product. With lot-to-lot consistency, reliable signal, and minimal noise, Molecular Probes® SAPE is an industry standard for Luminex® bead-based assays. Incorporate Molecular Probes® SAPE into your DNA or protein assay protocols to get more reliable and consistent results.

 

Molecular Probes® Streptavidin R-PE 
Molecular Probes® SAPE is consistent over a broad range of target concentrations.
Luminex® microspheres were incubated with 10 to 10,000 pg/mL TNF-alpha and detected with 4 μg/mL biotinylated detection antibody and 4 μg/mL of each reporter conjugate in a capture sandwich immunoassay. The relative net median fluorescence intensity achieved with each of the reporters is shown. Data are the averages from at least two independent experiments for each conjugate tested. Molecular Probes® SAPE shows superior consistency over the range tested.

DEPARTMENTS

On the Web

GeneAssist™ Pathway Atlas

GeneAssist™ Pathway Atlas for erythrocyte invasion by Plasmodium merozoites.

 

Find Interactive Pathway Maps at the GeneAssist™ Pathway Atlas

At our online GeneAssist™ Pathway Atlas, you’ll find more than 350 interactive pathway maps. Clicking on any protein in a pathway generates annotation data (protein names, gene names, transcripts, disease implications, etc.) and a list of Silencer® Select siRNAs, Silencer® siRNAs, and TaqMan® Assays that are appropriate for the study of that protein.

Imaging Corner

ProbesOnline Imaging Corner Enlarge Image  

Fluorescent Labeling of U2OS Cells

Live U2OS cells were transduced with CellLight® Mitochondria-GFP, BacMam 2.0 construct, then fixed and permeabilized. Cells were then treated with Anti–Green Fluorescent Protein (GFP), Rabbit Serum and labeled with Alexa Fluor® 594 Goat Anti–Rabbit IgG (red). The nucleus was stained with NucBlue® Fixed Cell Stain (blue). Mitochondria labeled with both CellLight® Mitochondria-GFP and Alexa Fluor® 594 Goat Anti-Rabbit IgG appear yellow. Cells were mounted in ProLong® Gold Antifade Reagent and imaged.

From the Bench

Fasting Increases the Efficacy of Chemotherapy in Cancer Cell Lines

Lee C, Raffaghello L, Brandhorstet S et al. (2012) Sci Transl Med 4:124ra27.

Long-term dietary restriction is shown to reduce oxidative stress and aging in many organisms, but would not be considered safe to use with cancer patients, many of whom are already struggling to maintain a healthy weight. In contrast, short-term starvation (48–60 hours of fasting) is also shown to protect mammalian cells from the effects of oxidative stress and chemotherapy agents, and would potentially not cause significant weight loss in patients undergoing such a treatment, making it a potentially useful augmentation to chemotherapy treatments. To investigate this, Lee and colleagues monitored several cancer cell lines and showed that cycles of starvation were as effective as chemotherapeutic agents in delaying progression of different tumors. Using the Click-iT® AHA protein synthesis assay, they were able to show that 4T1 tumor cells that were fasted showed a significant increase in translation and protein accumulation. The authors postulated that the increase in translation may result in the consumption of more energy by the cells, which may promote oxidative stress and cell death.

Molecular Probes® Free Webinars

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. Fluorophores are compounds that emit light at a specific wavelength when they have been excited at a lower wavelength. View the 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 will explore the basic characteristics, strengths, and weaknesses of various fluorophores to help you develop the best assay for your needs.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
© 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Cy is a registered trademark of GE Healthcare. Hoechst is a registered trademark of Hoechst GmbH. Zeiss and Axiovert are registered trademarks of Carl Zeiss AG Corporation. Luminex is a registered trademark of Luminex Corporation. Li-Cor and Odyssey are registered trademarks of Li-Cor, Inc. Feel free to distribute ProbesOnline™ newsletter to friends and colleagues, but please keep this copyright statement intact.