In This Issue


ReadyProbes® Reagents   Rapid, Convenient Cytoskeleton Labeling—ActinGreen™ 488 and ActinRed™ 555 ReadyProbes™ Reagents
Human Th1/Th2/Th17 Magnetic 8-Plex Panel   Measure Cytokines Produced by Th1/Th2/Th17 Lymphocytes—Human Th1/Th2/Th17 Magnetic 8-Plex Panel
Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B   Visualize Autophagosome-to-Autolysosome Progression in Living Cells—Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B
LysoTracker® Deep Red Reagent   New Near-Infrared Lysosomal Dye—LysoTracker® Deep Red Reagent
ABfinity™ Recombinant Antibodies   ABfinity™ Recombinant Antibodies—New Antibodies for Insulin
Qdot® Primary Antibody Conjugate   Qdot® Primary Antibody Conjugates for Flow Cytometry—New CD2, CD19, CD4, and CD45R Antibodies




Rapid, Convenient Cytoskeleton Labeling—ActinGreen™ 488 and ActinRed™ 555 ReadyProbes™ Reagents

What They Are
ActinGreen™ and ActinRed™ reagents are selective, high-affinity probes for F-actin, conjugated to some of our brightest and most robust fluorescent dyes. As with other ReadyProbes™ reagents, they are formulated as a room temperature–stable solution and come in convenient dropper bottles. Just tip and drip—two drops per milliliter to selectively stain the actin cytoskeleton.

What They Offer

  • High-affinity staining of F-actin with superior specificity compared to antibody methods
  • Ready-to-use liquid formulation in convenient dropper bottle—no need to dilute, weigh, or pipette
  • Stable at room temperature—keep handy at your bench or cell culture area

How They Work
Whether your interest in the cytoskeleton focuses on stem cell differentiation, tumor cell invasion, cell division, or using it as a marker in RNAi library screens or as a target in drug discovery, we offer a number of validated Molecular Probes® solutions for both live- and fixed-cell formats.

ActinGreen™ and ActinRed™ reagents are special formulations of our photostable and ultra-bright Alexa Fluor® 488 dye and Texas Red® dye conjugates of phalloidin. F-actin staining in fixed and permeabilized samples using dye–phalloidin conjugates outperforms antibody-based methods, and results using these probes have been featured in hundreds of publications. In the ReadyProbes™ format, they are now easier and more convenient to use than ever.

  • Learn more about ReadyProbes™ reagents
  • Find other cytoskeleton probes
  • Contact Technical Support for cytoskeleton staining protocols
ReadyProbes™ reagents  
Cytoskeleton labeling with ReadyProbes™ reagents. BPAE cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit. Actin was stained using ActinRed™ 555 ReadyProbes™ reagent, and nuclei were stained with NucBlue® Fixed Cell Stain.

Measure Cytokines Produced by Th1/Th2/Th17 Lymphocytes—Human Th1/Th2/Th17 Magnetic 8-Plex Panel

What It Is
The Human Th1/Th2/Th17 Magnetic 8-Plex Panel for the Luminex® platform is specifically designed for simultaneously quantifying 8 cytokines (IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17, and IFN-gamma) in serum, plasma, or tissue culture supernatant samples. Multiplexing provides more data from each sample, saving both money and time compared to ELISAs.

What It Offers

  • Get more from your precious samples—measure multiple proteins in the same sample
  • Magnetic beads—enable easy wash steps and facilitate automated washing protocols
  • Fast protocols—perform your assays and analyze your data typically in less than one day

How It Works
The assay has a 3.5-hour incubation time and is similar to an ELISA workflow. Magnetic microspheres are internally dyed with differing intensities, allowing differentiation of one bead from another. Beads covalently bound to different antibodies can be mixed in the same assay, utilizing a 96-well microplate format. At the completion of the sandwich immunoassay, beads can be read using any Luminex® system and xPONENT® software to distinguish bead color (analyte) and assay signal strength (PE) fluorescence intensity.

Luminex® Human Th1/Th2/Th17 Magnetic 8-Plex Panel assay  
Typical standard curves from the Luminex® Human Th1/Th2/Th17 Magnetic 8-Plex Panel assay, exhibiting large dynamic ranges.

Product Quantity Cat. No.
Human Th1/Th2/Th17 Magnetic 8-Plex Panel 100 tests LHC0015M

Visualize Autophagosome-to-Autolysosome Progression in Living Cells—Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B

What It Is
The Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B is a fluorescent protein–based biosensor that reports the formation of the autophagosome, followed by the maturation of the autophagosome as the lysosome fuses to it to form the autolysosome. This biosensor provides a more in-depth investigation of autophagy in live cells, over time.

What It Offers

  • More in-depth autophagy data—monitor the maturation of the autophagosome into the autolysosome
  • A complete solution—use in conjunction with LysoTracker® Deep Red to monitor the complete pathway of autophagy
  • Highly efficient—BacMam 2.0 technology enables autophagy to be studied across a broad range of cell types, including primary human cells, neurons, and stem cells

How It Works
The Premo™ Autophagy Tandem Sensor combines the autophagy marker LC3B with two fluorescent proteins, GFP and tagRFP. GFP is sensitive to low pH, whereas tagRFP is not. At the neutral pH of the autophagosome, fluorescence is observed from both GFP and tagRFP. Once the lysosome has fused, the pH drops, quenching GFP fluorescence and leaving only tagRFP fluorescence.


Premo™ Autophagy Tandem Sensor
Mechanism of action of the Premo™ Autophagy Tandem Sensor. (A)
Schematic representation of BacMam-mediated gene delivery and expression of the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B. (B) Validation of the Premo™ Autophagy Tandem Sensor as a biosensor to report changes in pH in LC3B-positive compartments. Treatment of cells with the vehicle causes no detectable accumulation of autophagosomes, resulting in a diffuse pattern of GFP and RFP fluorescence. Inhibition of autophagy using either chloroquine or leupeptin A results in the accumulation of LC3B-positive vesicles (autophagosomes/autolysosomes). Chloroquine neutralizes the pH of lysosomes, and therefore GFP and RFP fluorescence emission is observed. Leupeptin A doesn’t affect lysosomal pH, so only fluorescence from tagRFP is observed.

New Near-Infrared Lysosomal Dye—LysoTracker® Deep Red Reagent

What It Is
LysoTracker® Deep Red is a red-shifted, near-infrared probe designed to label lysosomes for fluorescence microscopy or flow cytometry.

What It Offers

  • Specifically detects lysosomes—monitored in the Cy®5 channel with peak Ex/Em of 647/668 nm
  • Easy multiplex capability—combine with green- or red-fluorescent reagents such as GFP and RFP
  • Complete solutions—use in conjunction with the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B to monitor the complete pathway of autophagy

How It Works
LysoTracker® Deep Red is a novel lysosomotropic agent that accumulates in acidic structures. In most cells these are limited to lysosomes. The excitation and emission properties of the LysoTracker® Deep Red reagent have been modified so that they exactly match the Cy®5 fluorescence channel, facilitating multiplex imaging with GFP and RFP.

LysoTracker® Deep Red Reagent
Labeling of lysosomes with LysoTracker® Deep Red. (A, B)
U2OS cells expressing CellLight® Lyso-GFP were labeled with 50 nM LysoTracker® Deep Red. LysoTracker® Deep Red shows colocalization with CellLight® Lyso-GFP. (C) Multicolor imaging using LysoTracker® Deep Red. HeLa cells expressing CellLight® Tubulin-GFP and CellLight® Late Endosomes-RFP were labeled with Hoechst 33342 and LysoTracker® Deep Red.

ABfinity™ Recombinant Antibodies—New Antibodies for Insulin

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.

Insulin is a hormone secreted by islet cells of the pancreas. It functions to regulate of blood glucose homeostasis, and modulate lipid metabolism. Insulin is a cleavage product of proinsulin produced by beta cells. Proinsulin is cleaved into three peptides. The A- and B-chains are covalently linked by disulfide bonds and make up the insulin molecule.

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 mixtures of recombinant monoclonal antibodies. These combine the improved signal strength that can come from using a polyclonal, with the highly reproducible results you get from ABfinity™ monoclonal antibodies.


ABfinity™ Recombinant Antibodies  
A human pancreatic tissue section was incubated with ABfinity™ Insulin Recombinant Rabbit Oligoclonal Antibody, and detection was achieved with green-fluorescent Alexa Fluor® 488 goat anti-rabbit antibody. Nuclei were stained with DAPI (blue).

Qdot® Primary Antibody Conjugates for Flow Cytometry—New CD2, CD19, CD4, and CD45R Antibodies

What They Are
Qdot® primary antibody conjugates of the mouse anti-human CD2 and CD19 markers and the rat anti-mouse CD4 and CD45R (B220) markers are now available in new colors for flow cytometry.

What They Offer

  • Compatibility—combine with existing organic dyes, increase the number of detectable parameters
  • Stability—do not degrade over time like tandem conjugates, afford greater reproducibility
  • Minimal single-laser compensation—narrow emission spectra allow for minimal compensation when using a single excitation source

How They Work
Qdot® particle primary antibody conjugates have extremely bright fluorescence emission that makes them well suited for the detection of low-abundance extracellular proteins. Efficient optical excitation is possible using the 405 nm violet excitation light source. In addition, the narrow, symmetric emission profiles of Qdot® particle conjugates require substantially lower compensation, enabling better, more efficient multicolor assays using the violet laser.


Qdot® Primary Antibody Conjugates  
Histogram overlay plot of gated human lymphocytes.
The black line represents cells labeled with CD2 Mouse Anti-Human mAb (clone S5.5) Qdot® 655 Conjugate, and the gray line represents unstained cells. Samples were acquired and analyzed using 405 nm excitation and a 660/20 bandpass emission filter using the Attune® Acoustic Focusing Cytometer (Blue/Violet option).



Fresh Tissue Staining of Glioma Cells in Mice

Glial cells, glioma cells of glial origin, and astrocytes express high levels of organic cation transporters and, unlike neurons, will rapidly accumulate ASP+, a fluorescent substrate for these transporters. Using a fresh tissue staining technique, Kucheryavykh and colleagues were able to observe stained cells within 30 minutes after ASP+ application. Their method allowed visualization of live glioma cells and tumor structures, which could easily be distinguished from stained astrocytes due to the morphological differences in these cells. Furthermore, even a 4-hour incubation in the stain did not appear to result in decreased viability in any of the glioma cell cultures tested.

Chemical structure of 4-Di-1-ASP  
Chemical structure of 4-Di-1-ASP (4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide).


Qdot® Conjugates Go the Distance to Monitor Lipid Raft Microdomains

Time-lapse imaging of live cells labeled with fluorescent dyes is a powerful technique for interrogating cell biology. As compared with organic fluorophores, the bright and photostable Qdot® conjugates can provide higher-content imaging by enabling more frequent image acquisitions with shorter exposure times. Additionally, our recently improved Qdot® conjugates now offer reduced intensity differences between the different colors, simplifying multiplex applications when using violet-light excitation.

Lipid Raft Labeling With Qdot® Conjugates

Lipid rafts are detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the plasma membrane. They have been shown to play a role in a variety of cellular processes, including compartmentalization of cell signaling events, regulation of apoptosis, and intracellular trafficking of certain membrane proteins and lipids, as well as in the infectious cycles of several viruses and bacterial pathogens [1–3].To demonstrate the advantages of Qdot® conjugates for time-lapse imaging, we labeled lipid rafts using either a direct Alexa Fluor® dye conjugate of cholera toxin subunit B (CT-B) or a biotinylated CT-B in conjunction with a Qdot® streptavidin conjugate [4].

Alexa Fluor® dye–labeled CT-B, a component of the Vybrant® Lipid Raft Labeling Kits, enables excellent endpoint and time-lapse imaging of lipid raft microdomains in the plasma membrane. However, when these probes are pushed to the limit—such as in time-lapse imaging with frequent, long, or intense exposures—photobleaching can occur. Alternatively, when biotinylated CT-B was labeled with a Qdot® 655 streptavidin conjugate, the signal remained bright during similar acquisition conditions.

Try Qdot® Conjugates in Place of Your Traditional Fluorophore Conjugates

Live-cell microscopy demands robust fluorophores that are bright, photostable, and multiplexable; these attributes are particularly important when imaging rare targets or highly dynamic cellular events such as membrane mobility and internalization processes. Qdot® particles meet these demands.

  1. Pike LJ (2009) J Lipid Res S323–S328.
  2. Simons K, Gerl MJ (2010) Nature Rev Mol Cell Bio 11:688–699.
  3. Lingwood D, Simons K (2010) Science 327:46–50.
  4. Chakraborty SK, Bruchez MP, Ballou B et al. (2007) Nano Lett 7:2618–2626.

Learn more about our broad selection of Qdot® conjugates and Qdot® labeling kits

Time-lapse imaging of lipid rafts in MMM murine macrophage cells
Time-lapse imaging of lipid rafts in MMM murine macrophage cells. (A)
Alexa Fluor® 594 dye–labeled cholera toxin subunit B (CT-B). (B) Qdot® 655 particle–labeled CT-B. Labeling experiments were performed at 4°C in complete medium. For Alexa Fluor® 594 labeling, we used the Vybrant® Alexa Fluor® 594 Lipid Raft Labeling Kit. For Qdot® 655 labeling, cells were incubated with 1 μg/mL biotinylated CT-B for 10 min followed by 10 nM Qdot® 655 streptavidin conjugate for 20 min and then a 1:200 dilution of anti–CT-B antibody (from the Vybrant® Lipid Raft Labeling Kit) for 15 min. Images were acquired at room temperature in Live Cell Imaging Solution. Alexa Fluor® 594 images were collected using 562/20 nm and 624/40 nm bandpass filters for excitation and emission, respectively. Qdot® 655 images were collected using a 435/20 nm bandpass excitation filter and a 515 nm longpass emission filter. Exposure times were adjusted to maximize the dynamic range of the fluorescent labeling.




On the Web

pHrodo indicators


pHrodo™ Indicators for Phagocytosis and Endocytosis

The proprietary pH-sensitive Molecular Probes® pHrodo™ dyes are almost nonfluorescent at neutral pH and fluoresce brightly in acidic environments. This increase in fluorescence signal at low pH makes them ideal for studying phagocytosis and endocytosis and its regulation by drugs or environmental factors. The minimal dye fluorescence at neutral pH also eliminates the need for wash steps and quencher dyes because any uninternalized dye will be essentially nonfluorescent. With both red and green pHrodo™ dyes available, you have more multiplexing opportunities as well as faster staining and more accurate results. Our website shows examples of applications in imaging, flow cytometry, and microplate assays.

Imaging Corner

Cytoskeletal Staining of Neonatal Fibroblasts

Gibco® Neonatal Human Dermal Fibroblasts were grown in Gibco®Medium 106 with Gibco® Low Serum Growth Supplement and plated on coverslips. Cells were fixed with 4% formaldehyde and permeablized with 0.2% Triton® X-100. Tubulin was labeled with anti–α-tubulin antibody and Molecular Probes® Alexa Fluor® 647 goat anti–mouse IgG (pink), F-actin was labeled with Molecular Probes® Alexa Fluor® 488 phalloidin (green), and nuclei were labeled with Molecular Probes® Hoechst 33342 (blue). Coverslips were mounted using Molecular Probes® ProLong® Gold Antifade Reagentand imaged on a Zeiss® LSM 710 confocal microscope. Image contributed by Kevin Chambers, Life Technologies Corporation.

From the Bench

Fluorescent Tracking Of Cardiac Cells Growing in Agarose Hydrogels

      Desroches BR, Zhang P, Choi BR et al. (2012) Am J Physiol Heart Circ Physiol 302(10):H2031–42.

Fluorescence-based cellular assays have yielded enormous insights into cellular structure and function over the past decades. However, the majority of cell models have employed cells adhered to a two-dimensional surface, e.g., a tissue culture dish. As cells in most tissues are arranged in a three-dimensional structure, the two-dimensional models may be lacking in physiological relevance: they may not provide the chemical, mechanical, and electrical stimuli cells experience during development and differentiation in vivo.

To provide models that better mirror the conditions in living organisms, intense efforts have been directed at developing three-dimensional (3D) tissue models that more faithfully reflect a true physiological environment. Many different approaches have been taken to create 3D cellular models, and a majority have relied on polymers or other scaffolds to promote tissue-like assemblies. Alternatively, scaffold-free approaches have been explored as well, and in this recent report a group from Brown University described a novel model of scaffold-free seeding of cardiac myocytes (CM) and fibroblasts (CF) to generate a 3D cardiac tissue model.

CM and CF cells were seeded in depressions in agarose hydrogels and allowed to self-assemble. The team found that the cells spontaneously formed viable and functional spherical microtissue that morphologically and functionally resembled cardiac tissue; tests included microelectrode recordings of action potentials, ion flux, cell viability, and membrane marker expression. Further, cell-type integrity in the microtissue was verified by adenoviral gene transfer.  Interestingly, the composition and function of the final spheroids was independent of whether CF and CM cells were seeded in the hydrogel wells simultaneously or sequentially, underscoring the inherent mechanisms that drive tissue formation. Such 3D tissue models hold great promise of supporting in vitro experiments that further our understanding of tissue development and function.

Reagents used in this investigation:

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