In This Issue


CellROX® Fluorescent Reagents   Oxidative Stress Detection—CellROX® Fluorescent Reagents
New Alexa Fluor® 790 Secondary Antibodies   Fluorescent Western Blotting—New Alexa Fluor® 790 Secondary Antibodies
Qdot® Streptavidin Conjugates   Smaller Pack Sizes for Cost-Effective Experiments—Qdot® Streptavidin Conjugates
VCP and PDI   Drama in the ER—Transitional Endoplasmic Reticulum ATPase (VCP) and Anti–Protein Disulfide Isomerase (PDI)
ABfinity™ Recombinant Antibodies   Tools for Analyzing Apolipoproteins—New ABfinity™ Recombinant Antibodies





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



XXVII Congress of the International Society for Advancement of Cytometry (CYTO 2012)
June 23–27, 2012
Congress Center Leipzig
Leipzig, Germany
Booth #233


Oxidative Stress Detection—CellROX® Fluorescent Reagents

what they are
CellROX® reagents are fluorogenic probes designed to reliably measure reactive oxygen species (ROS) in live cells. These cell-permeant reagents are nonfluorescent or very weakly fluorescent when in a reduced state but exhibit strong fluorescence upon oxidation. Now you can detect oxidative stress not only in the red channel but also in the orange and green channels with two new CellROX® reagents.

what they offer

  • Optimized probes for detection of oxidative stress in live cells
  • Simple 30-minute protocol and ability to multiplex with other compatible live-cell dyes
  • Compatibility with various platforms such as live-cell fluorescence imaging, high-content screening (HCS), flow cytometry, high-throughput screening (HTS), and the Tali®, FLoid™, and Attune® systems

how they work

Oxidative stress results from an imbalance between the production of ROS and the ability of cells to scavenge them. ROS oxidize DNA, proteins, and membrane lipids, damaging them in the process, and play an important role in the progression of many diseases. CellROX® reagents offer a simple live-cell workflow that can be adapted to various platforms and benchtop instruments, including the Tali® Image-Based Cytometer, FLoid™ Cell Imaging Station, and Attune® Acoustic Focusing Cytometer.


Human aortic smooth muscle cells  
Angiotensin II–induced oxidative stress in human aortic smooth muscle cells (HASMCs), measured with CellROX® Orange Reagent.
HASMCs were plated on glass-bottom 35 mm MaTtek dishes. Cells were left untreated (top) or treated with 500 nM angiotensin II for 4 hr at 37°C (bottom). Cells were then stained with 5 µM of CellROX™ Orange Reagent and NucBlue™ Live Cell Stain by adding the probes to the complete medium and incubating at 37°C for 30 min. Cells were washed with PBS and imaged on a Zeiss Axiovert inverted microscope using a 40x objective.

Absorption/Emission (nm) 644/665 545/565 485/520
Live-Cell–Compatible Yes Yes Yes
Formaldehyde-Fixable Yes No Yes
Detergent-Resistant No No Yes
Platforms* FC, HCS, HTS, I, Attune® system FC, HCS, I, Tali® system FC, HCS, HTS, I, and Tali®, FLoid™, and Attune® systems
Quantity 5 x 50 µL 5 x 50 µL 5 x 50 µL 1 kit
Cat. No. C10422 C10443 C10444 C10448
* FC = flow cytometry; HCS = high-content screening; HTS = high-throughput screening; I = imaging.

Fluorescent Western Blotting―New Alexa Fluor® 790 Secondary Antibodies

what they are
New Alexa Fluor® 790 secondary antibodies for fluorescent western blotting provide excellent signal-to-noise ratios on standard near-IR fluorescence scanners such as the LI-COR® Odyssey® Imaging System and the Kodak® image station.

what they offer

  • Bright, photostable fluorescence
  • Excellent signal-to-noise ratios
  • Sensitive detection

how they work

With Alexa Fluor® 680 and Alexa Fluor® 790 secondary antibodies, you can generate multicolor western blots that enable you to simultaneously evaluate multiple proteins on the same blot, even if the proteins comigrate. Multiplexing provides simple normalization and requires no blot stripping, no single-blot comparisons, and only two antibody-incubation steps, saving you time and sample. Alexa Fluor® 680 and Alexa Fluor® 790 secondary antibodies and streptavidin conjugates are ideal for fast and accurate multicolor western detection.


insulin-treated and untreated adipocytes   Induction of AKT (protein kinase B) phosphorylation in response to insulin treatment. Lysates of insulin-treated and untreated adipocytes were electrophoresed and transferred to nitrocellulose. Blots were incubated simultaneously with rabbit anti-AKT and mouse anti–phospho-AKT primary antibodies, then simultaneously with Alexa Fluor® 680 goat anti–mouse IgG and Alexa Fluor® 790 goat anti–rabbit IgG secondary antibodies. Total AKT protein bands are shown in red, and phospho-AKT bands are shown in green. Single-color images were overlaid; yellow bands indicate insulin-treated samples where phosphorylation of AKT occurs.
Product Alexa Fluor® 680 Alexa Fluor® 790
Streptavidin S32358 S11378
Goat anti–mouse IgG A21057 A11375
Goat anti–mouse IgG (H+L), highly cross-adsorbed A21058 A11357
Goat anti–rabbit IgG (H+L) A21076 A11367
Goat anti–rabbit IgG (H+L), highly cross-adsorbed A21109 A11369
Goat anti–rat IgG (H+L) A21096
Donkey anti–mouse IgG (H+L) A10038
Donkey anti–rabbit IgG (H+L) A10043 A11371
Donkey anti–goat IgG (H+L) A21084 A11370
Rabbit anti–mouse IgG (H+L) A21065
Rabbit anti–goat IgG (H+L) A21088

Smaller Pack Sizes for Cost-Effective Experiments—Qdot® Streptavidin Conjugates

what they are
Qdot® streptavidin conjugates are now available in cost-effective trial sizes. Pack sizes of 50 µL allow you to optimize your experiments and test these labels in novel applications.

what they offer

  • Sensitivity, brightness, and photostability
  • Efficiently excited with single-line excitation sources
  • Ideal for western blots, flow cytometry, fluorescence microscopy, and more

how they work

Qdot® streptavidin conjugates combine the robust sensitivity of biotin–streptavidin detection with the unparalleled brightness and photostability of Qdot® nanocrystals. Because streptavidin has a high binding affinity for biotin, Qdot® streptavidin conjugates can be used with biotin conjugates for sensitive, specific protein detection (e.g., to detect a biotinylated primary antibody bound to a protein target). These conjugates are compatible with diverse fluorescent applications, including flow cytometry, western blotting, and live- or fixed-cell imaging.


Lipid raft labeling with Qdot® streptavidin  

Lipid raft labeling with Qdot® streptavidin. MMM murine macrophage cells were labeled with cholera toxin subunit B biotin-XX conjugate followed by Qdot® 655 streptavidin conjugate. The cells were fixed, stained with Hoechst 33342, and imaged on a Zeiss® LSM confocal microscope.

Drama in the ER—Transitional Endoplasmic Reticulum ATPase (VCP) and Anti–Protein Disulfide Isomerase (PDI)

what they are
Anti-VCP mouse monoclonal IgG1,κ antibody binds to the 97 kDa ATPase necessary for mitotic fragmentation and reassembly of the Golgi apparatus and formation of transitional endoplasmic reticulum (tER) through vesicle budding. Anti–protein disulfide isomerase (PDI) antibodies, available as mouse IgG2b (SelectFX® kit) and recombinant rabbit monoclonal antibodies, bind to the ER-associated PDI.

what they offer

  • With an appropriate secondary antibody, anti-VCP antibody can be used for immunohistochemistry/immunocytochemistry, flow cytometry, imaging, immunoprecipitation, ELISAs, and in-cell ELISAs
  • The SelectFX® Alexa Fluor® 488 Endoplasmic Reticulum Labeling Kit is a complete imaging kit that provides the anti-PDI primary antibody, Alexa Fluor® 488 goat anti-mouse secondary antibody, fixative and permeabilizing solution, blocking solution, and PBS
  • Anti-PDI (ER marker) recombinant antibodies are validated for western blotting

how they work

Localized mostly in the cytoplasm and nucleus, VCP forms a ternary complex with other proteins that either promotes or inhibits Golgi membrane fusion and endoplasmic reticulum biogenesis. Defects in VCP are involved in amyotrophic lateral sclerosis (ALS) and Parkinson’s disease.

PDI is a multifunctional enzyme that catalyzes disulfide bond formation, reduction, or isomerization of proteins in the lumen of the ER and mediates the correct folding of newly synthesized proteins.

Tools for Analyzing Apolipoproteins―New ABfinity™ Recombinant Antibodies

what they are
Apolipoprotein A1 (Apo A1) is the major constituent of human high-density lipoprotein (HDL) and plays an important role in metabolism and transport of lipoproteins in serum. The protein acts as an activator for lecithin cholesterol acyl transferase, which converts cholesterol to cholesteryl esters. Apolipoprotein A2 (Apo A2), another important component of human HDL, is involved in the hepatic lipase reaction. Like Apo A1, Apo A2 has the ability to take up cholesterol and phospholipids from cell membranes. However, Apo A2 does not undergo the changes observed with Apo A1 in the presence of lecithin cholesterol acyl transferase. Apolipoprotein E (Apo E) plays an important role in the formation of very low density lipoprotein and chylomicrons. Apo E is involved in distributing cholesterol, which is available in excess in certain cells. Several new ABfinity™ recombinant antibodies for Apo A1, Apo A2, and Apo E are available.

what they offer

  • Consistent lot-to-lot antibody performance
  • Minimizes 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. This production process provides consistent antibodies from lot to lot. ABfinity™ oligoclonal antibodies are a mixture of recombinant monoclonal antibodies, combining the improved signal strength of a polyclonal with the highly reproducible results you get from ABfinity™ monoclonal antibodies.


HepG2 cells stained with Apo E ABfinity™ Recombinant Rabbit Oligoclonal Antibody  
Immunocytochemistry analysis of HepG2 cells treated with Apo E ABfinity™ Recombinant Rabbit Oligoclonal Antibody. (A) Alexa Fluor® 488 goat anti-rabbit was used as the secondary antibody (green). (B) Nuclei were stained with DAPI (blue). (C) Actin was stained with Alexa Fluor® 594 phalloidin (red). (D) Composite image of cells showing cytoplasmic localization of Apo E.



Selectively Labeling Arteries for Neurovascular Studies

A Novel Use for Alexa Fluor® 633 Hydrazide

In a recent publication to analyze how active neurons regulate blood flow in surrounding vessels, Shen et al. demonstrated the utility of Alexa Fluor® 633 hydrazide for selectively labeling the neocortical arteries of rodents and cats in vivo. Whether Alexa Fluor® 633 hydrazide was delivered in vivo (by intravenous injection or locally to the cerebral cortex by patch pipette) or applied to tissue sections, the authors observed that arteries and arterioles exhibited extremely bright fluorescent labeling while veins, venules, and microvessels remained unlabeled. In addition, only Alexa Fluor® 633 hydrazide––labeled vessel walls showed stimulus-evoked dilation consistent with the known dilation dynamics of arterioles. Perhaps most importantly, by using Alexa Fluor® 633 hydrazide to selectively label arterioles, Shen and coworkers discovered that arteriole dilation decreased the fluorescence of neurons located directly beneath those vessels, while the fluorescence of neurons directly below veins was unaffected. Thus, artery wall labeling in vivo with Alexa Fluor® 633 hydrazide can be used to identify dips in neuronal fluorescence caused by arteriole dilation―a potential artifact in neuronal imaging experiments. This extends the potential utility of Alexa Fluor® 633 hydrazide to the study of arteriole dilation dynamics, allowing high-resolution in vivo monitoring of vessel dilation and constriction in brain and other vascularized tissues.



Bright, Photostable Qdot® Nanocrystal Streptavidin Conjugates

When you combine bright and photostable Qdot® nanocrystals with the high binding affinity of streptavidin for biotin, you experience superior signal intensity coupled with an increase in specificity. Qdot® streptavidin conjugates consist of a biotin-binding protein (streptavidin) covalently attached to a fluorescent label (Qdot® nanocrystal). Most dye conjugates are synthesized by attaching one or more fluorophores to a single biomolecule; however, the large surface area afforded by the nanocrystal allows simultaneous conjugation of many biomolecules to a single Qdot® nanocrystal. The advantages of this approach include increased avidity for targets, the potential for cooperative binding, and the use of efficient signal amplification methodologies. For example, combining biotin-functionalized products with streptavidin labels allows for successive enhancement in signal via "sandwiching" (streptavidin/biotin/streptavidin, etc.) following an initial labeling step. Qdot® bioconjugates can be detected using a standard fluorescence microscope. Although these reagents are optimally excited using the 405 nm violet laser, they can efficiently absorb white light when used with broad-excitation filters.


Live human osteosarcoma cells   Fixed-cell immunocytochemistry labeling and detection. Human osteosarcoma cells were fixed, permeabilized, and blocked using the Endogenous Biotin-Blocking Kit.  Mitochondria were labeled with an Anti–ATP Synthase Subunit β Monoclonal Antibody , biotinylated with Biotin-XX Goat Anti–Mouse IgG, and detected with Qdot® 655 Streptavidin Conjugate. The nucleus was labeled with green-fluorescent SYTOX® Green Nucleic Acid Stain. Cells were mounted in Qmount® Qdot® Mounting Medium and imaged on a standard wide-field epifluorescent microscope.



On the Web

Tali® Image-Based Cytometer


Discover the Tali® Image-Based Cytometer

The tools to begin your discovery of the Tali® Image-Based Cytometer are just a click away. From the Tali® web page you can review recent application data and dye compatibility results. You’ll also find links to videos, FAQs, and software and firmware updates.

Imaging Corner

Imaging Corner
Click to enlarge

Click Chemistry Labeling of Tetrahymena

Live Tetrahymena pyriformis were cultured with 5-ethynyl-2′-deoxyuridine (EdU), tetraacetylated N-azidoacetylgalactosamine (Click-iT® GalNAz Metabolic Glycoprotein Labeling Reagent) and microspheres from the InSpeck™ Blue (350/440) Microscope Image Intensity Calibration Kit (2.5 µm, 10% intensity). Cells were fixed and permeabilized. Using the Cu(I)-mediated click reaction, EdU incorporated into the DNA was labeled with Alexa Fluor® 488 azide, and GalNAz incorporated into the cellular components was labeled with Alexa Fluor® 555 alkyne. After the click reaction, cilia were labeled with mouse anti–β-tubulin antibody and Alexa Fluor® 647 goat anti–mouse IgG and mounted in SlowFade® Gold Antifade Reagent. Image contributed by Kary L. Oakleaf and Olivia Cholewa, Life Technologies.

From the Bench

Caught Green-Handed—Fingerprinting Using Fluorescent Dyes

Hazarika P, Russell DA (2012) Angew Chem Int Ed 51:3524–3531.

Fingerprint analysis has been a critical technique for human identification in forensic investigations since the late 19th century, and methods currently in use typically involve development of an invisible print using chemical or physical treatments. In a recent publication, Hazarika and Russell demonstrated the utility of gold nanoparticles and magnetic particles functionalized using antibodies against cotinine, THC, methadone and its metabolite EDDP, and benzoylecgonine (metabolite of cocaine) in detecting these substances in human fingerprints. Briefly, latent fingerprints were first treated with the functionalized particles, and the pattern of particle binding was detected using Alexa Fluor® dye–labeled antibodies. The authors demonstrated that the technique was able to specifically detect the various illicit substances (no signal on fingerprints deposited by non–drug users) and that multiple drug metabolites can be detected simultaneously in the same fingerprint. Because the technique produces images of high evidential quality, the authors hope that modifications to make this approach more portable will allow it to become commonplace at sites of forensic investigation.

Molecular Probes® Webinars

Molecular Probes Webinar Series


Flow Cytometry in Microbiological Research

The application of flow cytometry to microbiological research has expanded from detection and quantification of organisms to more complex studies, including analysis of host–microbe interactions and detailed spatial and temporal analyses of microbial metabolism in different environments. In this webinar we’ll discuss how the multiparametric nature of flow cytometry can be applied to microbiology, and the advantages of using this application over traditional microbiological methods.

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