ProbesOnline—A Molecular Probes Publication

In This Issue


CellEvent™ and CellROX™ Reagents   Multiplex Detection of Apoptosis and Oxidative Stress — CellEvent™ and CellROX™ Reagents
Recombinant Osteonectin Antibodies   Study Osteonectin Expression With New Recombinant Antibodies — Osteonectin Monoclonal and Oligoclonal Antibodies




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Multiplex Detection of Apoptosis and Oxidative Stress—CellEvent™ and CellROX™ Reagents

what they are
The CellEvent™ Caspase-3/7 Green Detection Reagent is a new reagent for imaging apoptosis that does not require any wash steps. The CellROX™ Deep Red Reagent is a novel fluorogenic probe for detecting oxidative stress. When used together, these reagents can be used to simultaneously detect reactive oxygen species (ROS) and apoptosis in live or fixed cells.

what they offer
  • Ultrasensitive and specific reagents to detect oxidative stress and apoptosis
  • Compatible with formaldehyde-based fixation methods
  • Compatible with traditional microscopy, flow cytometry, high-content screening, and high-throughput screening
how they work
To detect apoptosis, the CellEvent™ Caspase-3/7 Green substrate emits fluorescence in the presence of activated caspase-3/7, a hallmark of programmed cell death. The cell-permeant CellROX™ dye is nonfluorescent in a reduced state and exhibits bright fluorescence upon oxidation by ROS.

CellROX™ Deep Red Reagent and CellEvent™ Caspase-3/7 Detection Reagent

Fluorescence imaging of oxidative stress and apoptosis using CellROX™ Deep Red Reagent and CellEvent™ Caspase-3/7 Green Detection Reagent.

HeLa cells were treated with or without 0.5 µM staurosporine for 2 or 4 hr in the presence of 7.5 µM CellEvent™ reagent. Cells were then stained with 5 µM CellROX™ reagent and Hoechst 33342 for 30 min at 37°C, then washed with warm DPBS. Cells were imaged immediately on a Zeiss Axiovert® inverted microscope using a 40x objective. Oxidative stress was observed at 2 hr after treatment (magenta), while caspase-3/7 activation was not observed until 4 hr after treatment (green).

Study Osteonectin Expression With New Recombinant Antibodies—Osteonectin Monoclonal and Oligoclonal Antibodies

what they are
Osteonectin (SPARC) is a bone-related marker protein secreted by osteoblasts and activated during osteogenic differentiation. Overexpression of osteonectin is observed in many cancers, making osteonectin an important research target and potential target for treatment. We now offer highly specific recombinant monoclonal and oligoclonal antibodies for osteonectin.

what they offer

  • Consistent results
  • Exceptional sensitivity and specificity
  • Available in monoclonal (single clone) or oligoclonal (pool of clones) format


how they work
Osteonectin antibodies are available as ABfinity™ Recombinant Rabbit Monoclonal and Recombinant Rabbit Oligoclonal Antibodies. This recombinant technology helps to ensure consistent antibody performance lot after lot, so you don’t have to revalidate dilutions for your experiments when you order more. These antibodies are validated for western blotting applications.

Western blot detection of Osteonectin with Osteonectin Recombinant Rabbit Oligoclonal Antibody

Western blot detection of osteonectin with Osteonectin Recombinant Rabbit Oligoclonal Antibody.


New Technique for Measuring Oxidative Stress

The oxidation state of protein thiols can be used to indicate oxidative stress levels in cells and tissues. However, quantitative detection of protein thiol oxidation state in tissue samples poses several technical barriers, including the ability to prepare samples without affecting oxidation state as well as to detect relatively subtle changes. A new fluorescence technique for measuring protein thiol oxidation state that overcomes these challenges was recently described in the literature [1], enabling accurate and reliable detection of subtle changes in the global protein thiol oxidation state by microplate fluorometry, and subsequently in the oxidation state of specific proteins by SDS-PAGE.  

Briefly, Armstrong et al. describe techniques developed to improve protein solubilization while preventing artifactual protein oxidation of the sample. During the solubilization step, reduced thiols are labeled with the green-fluorescent BODIPY® FL maleimide (FLm). Following removal of excess FLm, remaining oxidized protein thiols are reduced with TCEP. The newly reduced protein thiols are then reacted with the red-fluorescent Texas Red® maleimide (TRm), after which the excess reactive TRm is removed. 

Because the BODIPY® FL and Texas Red® fluorophores exhibit minimal fluorescence emission overlap, protein labeling with these two fluorescent dyes allows quantitative measurement of both reduced and oxidized protein thiols in the same sample using a standard fluorescence microplate reader. By measuring both oxidized and reduced protein thiols, researchers can detect small differences in biological samples that might be missed using techniques that only quantitate reduced thiols, such as reaction with Ellman’s reagent. Furthermore, this dual labeling technique allows changes in the thiol oxidation state of specific proteins to be visualized by analyzing samples using SDS-PAGE and a fluorescent gel scanner.

  1. Armstrong A, Zerbes R, Fourier P, Arthur P (2011) A fluorescent dual labeling technique for the quantitative measurement of reduced and oxidized protein thiols in tissue samples. Free Radical Biology & Medicine 50:510–518.

  BODIPY® FL N-(2-aminoethyl)maleimide (A) and Texas Red® C2 maleimide (B).

BODIPY® FL N-(2-aminoethyl)maleimide (A) and Texas Red® C2 maleimide (B).


Reagents for Studying the Cytoskeleton in Live and Fixed Cells

The cytoskeleton invokes images of rigidity and immobility. And while the cytoskeleton does provide a cellular scaffold, supports cell junctions, and imparts intracellular compartmentalization, it also is a key component in several important highly dynamic processes: intracellular organelle transport, cell division, motility, and signaling. The key role of the cytoskeleton in cell division and cell motility makes it central to not only normal but also malignant processes (unchecked proliferation and invasiveness); cytoskeletal components are targets of several cancer and anti-inflammatory drugs.

Whether you’re studying the cytoskeleton in the context of stem cell differentiation, cancer biology, as a marker in RNAi library screens, or as a target in drug discovery, Molecular Probes® cytoskeletal reagents offer validated solutions:

  • Superior performance in live- or fixed-cell analysis
  • Highly specific staining with the brightest fluorophores
  • Validated in hundreds of publications

Alexa Fluor® Phalloidin Conjugates
Our photostable and ultrabright Alexa Fluor® phalloidin conjugates selectively stain F-actin, yielding precise results. Phalloidin conjugates are available with all the industry-leading Alexa Fluor® dyes and are optimal for fixed and permeabilized samples. Some recent examples of the use of Alexa Fluor® phalloidin conjugates in cancer-related research are cited in the references below [1–7].

TubulinTracker™ and CellLight® Reagents
TubulinTracker™ and CellLight® reagents are ideal for capturing cytoskeletal dynamics and processes in live cells. TubulinTracker™ Green, a fluorescent derivative of taxol (paclitaxel), provides green-fluorescent staining of polymerized tubulin in live cells. CellLight® reagents are fusion constructs of actin, tubulin, or MAP4 with emGFP or TagRFP. Achieve highly efficient expression even in sensitive cells such as stem cells, neurons, and primary cells—just add the CellLight® reagent to cells in complete medium, incubate, and image the next day. CellLight® reagents are easy to use in multiplexed fixed-cell analyses with antibodies after formaldehyde treatment.

Antibodies to Cytoskeletal Proteins
Invitrogen™ antibodies span multiple steps in the process of cell migration, which is controlled by signaling events cascaded through the cytoskeleton. Adhesion molecules are critical in this process, and timing and regulation of signaling events are mediated by many site-specific phosphorylation events. Our antibodies can be used in ICC applications in conjunction with many of our fluorescent protein markers to study cellular morphology.


SelectFX® Nuclear Labeling Kit and Alexa Fluor® phalloidin conjugates

Fixed, permeabilized bovine pulmonary artery endothelial cells visualized using components of the SelectFX® Nuclear Labeling Kit and Alexa Fluor® phalloidin conjugates.


  1. Wu M, Pastor-Pareja JC, Xu T (2010) Nature 463:545–548.
  2. Lee K, Gallop JL, Rambani K et al. (2010) Science 329:1341–1345.
  3. Nakamura S, Kobayashi K, Nishimura T et al. (2010) Science 328:1561–1563.
  4. Gong H, Shen B, Flevaris P et al. (2010) Science 327:340.
  5. West JA, Viswanathan SR, Yabuuchi A et al. (2009) Nature 460:909–913.
  6. Takeda A, Baffi JZ, Kleinman ME et al. (2009) Nature 460:225–230.
  7. Han HJ, Russo J, Kohwi Y, Kohwi-Shigematsu T (2008) Nature 452:187–193.


On the Web

Molecular Probes® CELLImaging Guide  

Imaging Reagent Selection — There’s an App for That

Introducing the new Molecular Probes® CELLImaging Guide with protocols. From your iPhone®, now you can:

  • Select reagents by application area
  • Visualize predictive results for each reagent
  • View streamlined, intuitive protocols
  • Track protocol progress with our built-in timer

Apps compatible with iPad® and Droid coming soon!


The View

Stimulated emission depletion (STED) microscopy of tubulin in live Kyoto cells visualized by Tubulin Tracker™ Green

Stimulated emission depletion (STED) microscopy of tubulin in live Kyoto cells visualized with TubulinTracker™ Green.

Confocal (left) and STED (right) images were acquired with a Leica TCS STED CW microscope. Images were pseudocolored red. Images contributed by Jochen Sieber, Leica Microsystems.


Product Quantity Cat. No.  
TubulinTracker™ Green 1 set T34075 Order Now


Improving Drug Potency and Efficacy by Nanocarrier-Mediated Subcellular Targeting

Murakami M, Cabral H, Matsumoto Y et al. (2011) Sci Transl Med 3(64):64ra2.

In the fight against cancer, researchers work to develop strategies to overcome many obstacles to effective drug therapies, including nontargeted drug application and multidrug resistance of some cancer cell types. Murakami and colleagues have investigated the delivery of anticancer compounds using nanocarriers (containing the fluorescent labels BODIPY® TR SE and BODIPY® FL hydrazide). Real-time cellular uptake, intracellular trafficking, and drug release were measured by fluorescence (using the probes LysoTracker® Blue, CellLight™ Early Endosomes-RPF, and CellMask™ Deep Red Plasma Membrane Stain); the effect on drug efficacy by subcellular pathways was similarly investigated. The in vivo behavior of the nanocarrier was studied in a mouse xenograft model using time-lapse confocal laser-scanning microscopy. It was demonstrated that the nanocarrier remained in circulation for at least 12 hr, with concomitant accumulation in tumor cells. Release of the active drug inside tumor cells was measured beginning at 4 hr post-injection. IC50 measurements indicated that nanocarrier-mediated drug delivery was more effective than the free drug, which the authors hypothesized was due to a reduction in intracellular adduct formation through more direct delivery to the nucleus. The authors further speculated that adoption of this therapeutic technique may reduce the incidence of acquired drug resistance.

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