In This Issue


CellLight® Actin-RFP BacMam 2.0   Visualize Actin Before Fixing Your Cells—CellLight® Actin-RFP BacMam 2.0
  New Antibody for Ocular Development Studies—PiTX3 ABfinity™ Recombinant Rabbit Monoclonal Antibody




The Dirty Lab Coat blog Customer Spotlight: The Dirty Lab Coat
Cornell University student Rudy Nazitto blogs about interesting science and experimental data. Look for reviews of Life Technologies products, including the Qubit® 2.0 Fluorometer.


Visualize Actin Before Fixing Your Cells—CellLight® Actin-RFP BacMam 2.0

what it is
CellLight® Actin-RFP BacMam 2.0 reagent is a modified insect virus (baculovirus) containing an actin–Red Fluorescent Protein (RFP) fusion construct. With this probe, you can visualize actin in live cells, then fix your cells and amplify the fluorescent signal using a highly specific anti-RFP antibody. This flexibility offers an advantage over labeling with traditional actin probes such as conjugated phalloidin, which require that cells be fixed with methanol, making live-cell imaging impossible. In addition, phalloidin is not multiplexable with other dyes and antibodies for which formaldehyde fixation is preferred.

what it offers

  • Highly efficient—>90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
  • Fast and convenient—simply add, incubate overnight, and image, or store frozen, assay-ready cells for later use
  • Robust—nonreplicating in mammalian cells, lacking observable cytopathic effects, and suitable for biosafety level (BSL) 1 handling
  • Flexible—co-transduce more than one BacMam reagent for multiplex experiments or co-localization studies, and tightly control expression levels just by varying the dose

how it works

CellLight® reagents combine the utility and selectivity of fluorescent proteins with the transduction efficiency of BacMam technology, enabling unambiguous staining of cellular structures and processes in live mammalian cells. Provided in ready-to-use format—simply add, incubate, and image—CellLight® reagents provide highly efficient expression in cell lines, primary cells, stem cells, and neurons.


CellLight® Actin-RFP BacMam 2.0  
Actin (red) and talin (green) visualized in human neonatal epidermal keratinocytes. Cells were co-transduced with CellLight® Actin-RFP and CellLight® Talin-GFP and imaged on a Zeiss LSM confocal microscope after overnight incubation.

New Antibody for Ocular Development Studies—PiTX3 ABfinity™ Recombinant Rabbit Monoclonal Antibody

what it is
Pituitary homeobox 3 (PiTX3) is a member of the RIEG/PiTX homeobox family, which also includes the DNA-binding proteins PiTX1 and PiTX2. PiTX3 is involved in lens formation in early ocular development and is later expressed in other organs and limbs. This protein has a highly restricted expression pattern in the brain. Mutations of the PiTX3 gene have been associated with abnormalities in ocular structure such as anterior segment dysgenesis (ASD) and a familial form of congenital cataracts.

what it offers

  • Superior lot-to-lot consistency, minimizing the need to revalidate working antibody dilutions each time you order
  • Extremely high specificity and sensitivity
  • Validation in western blotting and indirect ELISA

how it works

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.


PiTX3 ABfinity™ Recombinant Rabbit Monoclonal Antibody  
Immunocytochemical analysis of U2OS cells. (A) Cells were treated with PiTX3 ABfinity™ Recombinant Rabbit Monoclonal Antibody (5H10L5) and labeled with Alexa Fluor® 488 goat anti–rabbit IgG (green). (B) Nuclei were stained with DAPI (blue). (C) Glycoconjugates on the cell membrane were stained with Alexa Fluor® 594 wheat germ agglutinin (red). (D) Composite image of cells showing nuclear localization of PiTX3.



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. To demonstrate the advantages of Qdot® conjugates in time-lapse imaging, we labeled lipid rafts using either an Alexa Fluor® dye conjugate of cholera toxin subunit B (CT-B) or a biotinylated CT-B in conjunction with a Qdot® streptavidin conjugate. 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 of 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. In contrast, when biotinylated CT-B was labeled with a Qdot® 655 streptavidin conjugate, the signal remained bright during similar acquisition conditions.

Live-cell microscopy (and especially imaging of rare targets or dynamic cellular events) requires bright, photostable, and multiplexable fluorophores. In comparison to organic fluorophores, Qdot® conjugates can provide higher-content imaging by enabling more frequent image acquisitions with shorter exposure times.


Time-lapse imaging of lipid rafts in MMM murine macrophage cells
Time-lapse imaging of lipid rafts in MMM murine macrophage cells. (A)
Cells labeled with Alexa Fluor® 594 dye–labeled cholera toxin subunit B (CT-B). (B) Cells labeled with Qdot® 655 nanocrystal–labeled CT-B. Labeling experiments were performed at 4°C in complete medium. For Alexa Fluor® 594 labeling, the Vybrant® Alexa Fluor® 594 Lipid Raft Labeling Kit was used. 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, used to crosslink the CT-B–labeled rafts into distinct patches that are easily visualized) 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.



A Diverse Selection of Organelle Probes

Life Technologies offers a wide array of cell-permeant fluorescent stains that selectively associate with live-cell structures. These stains are powerful tools for investigating respiration, mitosis, apoptosis, multidrug resistance, substrate degradation and detoxification, intracellular transport, sorting, and much more. With a broad selection of fluorescent colors available, researchers can perform multiplex labeling of a variety of organelles, including mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus, as well as subcellular structures such as actin filaments and microtubules, and even the plasma membrane, in live mammalian cells.

These probes, which include our MitoTracker®, LysoTracker®, LysoSensor™, CellMask™, RedoxSensor™, and ER-Tracker™ organelle stains, are compatible with most fluorescence instrumentation and can be used to investigate organelle structure and activity with minimal disruption of cellular function.


Organelle Probes  
Live-cell labeling using a selection of organelle stains. Live human osteosarcoma cells were labeled with CellMask™ Deep Red Plasma Membrane Stain, the endoplasmic reticulum–selective ER-Tracker™ Green Dye (BODIPY® FL Glibenclamide), mitochondrion-selective MitoTracker® Red CMXRos Dye, and blue-fluorescent NucBlue™ Live Cell Stain (Hoechst 33342 special formulation). Labeled cells were imaged in Live Cell Imaging Solution on the Zeiss 710 point-scanning spectral detection confocal microscope.



On the Web

Flow Cytometry Resources


Flow Cytometry Resources at Your Fingertips

Now you can find all flow cytometry resources in one place. Access free flow cytometry tutorials and educational webinars, download the flow cytometry reagent mobile app, read the latest publications and literature, and learn tips and tricks to optimize your flow cytometry experiments.

Imaging Corner

Imaging Corner Enlarge Image  

Dual Labeling of ManNAz-Modified Proteins and Protein Synthesis

To label ManNAz-modified proteins, U2OS cells were cultured in McCoy’s medium containing 10% FBS and 50 µM ManNAz for 24 hr. Cells were then cultured for 1 hr in McCoy’s medium containing 10% FBS and 50 µM HPG to label all newly synthesized proteins. Cells were fixed and permeabilized before being labeled with serial click reactions using the Click-iT® Cell Reaction Buffer Kit. Alexa Fluor® 488 alkyne was used to label ManNAz-modified proteins, and Alexa Fluor® 647 azide was used to detect HPG incorporation. Image contributed by Nicholas Dolman, Life Technologies.

From the Bench

Glycan Metabolic Labeling of E. coli Using Click Chemistry

Dumont A, Malleron A, Awwad M, Dukan S, Vauzeilles B (2012) Angew Chem Int Ed 51:3143–3146.

Gram-negative bacteria such as E. coli are covered in a dense layer of lipopolysaccharide, and these molecules add to the structural integrity of the organism and are also responsible for pathogenicity in many strains. In the past, researchers have attempted glycan metabolic labeling of E. coli, but have succeeded only in a genetically engineered strain. In new research, Dumont and colleagues have targeted a specific sugar (3-deoxy-D-manno-octulosonic acid (KDO)) present in almost all gram-negative bacteria. By incubating E. coli with a KDO analog (8-azido-8-deoxy-KDO) overnight, they were able to use azide-alkyne click chemistry to specifically attach a fluorescent dye to the surface. After 5 minutes in the presence of the labeling reagent, the surface was brightly labeled. Because the KDO analog contains a modification at the C8 position, reverse metabolism (and dissemination of the chemical reporter into other carbohydrates) should be minimal. The authors postulate that this labeling technique has potential applications in bacterial cell imaging, prodrug conjugation, and direct drug delivery.

Molecular Probes® Webinar Series

Molecular Probes Webinar Series


Free Webinar—The Meaning of Life at the Cellular Level: Detecting Apoptosis With Fluorescence

Induction of apoptosis is a key cellular regulator in both development and disease. Apoptosis is a multifaceted process involving multiple cellular effectors. Life Technologies offers a number of solutions for researchers using fluorescent probes to study key components of the apoptotic machinery. This webinar will:

  • Provide an overview of the features of apoptosis
  • Describe key parameters that can be measured to assay apoptosis
  • Offer a comprehensive guide to available labeling and detection technologies for apoptosis research
  • Provide tips and tricks on how to best implement those technologies

Choose from two different times—both will be presented live.

Date: Thursday, May 24, 2012
7:00 a.m. PDT or 11:00 a.m. PDT

© 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Feel free to distribute ProbesOnline to friends and colleagues, but please keep this copyright statement intact.