In This Issue


LCIS   The Clear Imaging Choice—Live Cell Imaging Solution
NucBlue™   Live- and Fixed-Cell Nuclear Solutions—NucBlue™ Live and Fixed Cell Stains
Alkaline Phosphatase Live Stain   Identify Pluripotent Colonies Without Killing Cells—New Alkaline Phosphatase Live Stain
ABfinity™ Recombinant Antibodies   Study Nucleostemin With a New Recombinant Antibody—ABfinity™ Recombinant Antibody
CellLight® Reagents   New FP-Based Labels for Key Cellular Targets—CellLight® Reagents



BioProbes® Journal of Cell Biology Applications
BioProbes 66  
The Molecular Probes® Handbook
Molecular Probes Handbook


The Clear Imaging Choice—Live Cell Imaging Solution

what it is
The Molecular Probes® Live Cell Imaging Solution is a physiological medium developed for live-cell imaging, dye loading, and wash steps. This reagent is an optically clear solution buffered with HEPES at pH 7.4 that keeps cells healthy for up to 4 hours at ambient atmosphere and temperature.

what it offers

  • Physiological and chemical optimization—the ideal solution for live-cell fluorescence imaging applications
  • Optical clarity―affording more efficient imaging and minimal background
  • Extended buffering capacity compared to other media―providing improved cell viability

how it works

For reliable results in live-cell imaging, cells must be maintained as closely as possible to physiological temperature, pH, oxygen tension, and other conditions. Live Cell Imaging Solution provides better clarity, signal-to-noise ratio, and cell viability than standard media. In addition, the physiological composition is based on Ringer’s solution, making Live Cell Imaging Solution ideal for other cell-based applications such as wash steps and dye loading.


Live Cell Imaging Solution  
Comparison of live cells imaged in Live Cell Imaging Solution and another medium. CHO-M1 cells were dye-loaded and imaged in Live Cell Imaging Solution (LCIS) or complete F-12/Hamm’s medium with 10% FBS.
Product Quantity Cat. No.
Live Cell Imaging Solution 500 mL A14291DJ

Live- and Fixed-Cell Nuclear Solutions—NucBlue™ Live and Fixed Cell Stains

what they are
Hoechst 33342 and DAPI are popular cell-permeant nuclear counterstains that emit blue fluorescence when bound to DNA. We have reformulated these classic stains as room temperature–stable solutions—NucBlue™ Live Cell Stain and NucBlue™ Fixed Cell Stain—that are provided in convenient dropper bottles. Just use two drops per milliliter to stain your cells.

what they offer

  • Ready-to-use liquid formulations of room temperature–stabilized, high-purity Hoechst 33342 and DAPI
  • Apply 2 drops/mL—no need to dilute, weigh, or pipet
  • Convenient dropper bottle to keep at your microscope or cell culture area

how they work

Hoechst 33342 is perhaps the most common fluorescent nuclear stain for live and fixed cells and tissue sections. Similarly, DAPI is a classic fluorescent dye used extensively for nuclear staining of fixed cells. These reagents are typically supplied as solids that must be weighed out, or as highly concentrated solutions that must be diluted several thousand–fold before use. The recommended storage is generally in a freezer or refrigerator. In contrast, the NucBlue™ Live Cell Stain and NucBlue™ Fixed Cell Stain formulations of these imaging reagents are provided ready to use and can be stored right next to your microscope or workstation. Packaged in ultraconvenient dropper bottles, NucBlue™ reagents enable you to counterstain your cells whenever and wherever they are ready.


NucBlue™ Fixed Cell Stain  

Endothelial cells stained with ready-to-use NucBlue™ stain. Bovine pulmonary artery endothelial cells (BPAEC) stained with NucBlue™ Fixed Cell Stain, BODIPY® FL Phallacidin, and MitoTracker™ Red CMXRos and imaged on the FLoid™ Cell Imaging Station.

Identify Pluripotent Colonies Without Killing Cells―New Alkaline Phosphatase Live Stain

what it is
Screen for pluripotency without sacrificing precious colonies, using the only alkaline phosphatase stain that maintains cell viability. With this new nontoxic stain, you can easily label pluripotent stem cells and monitor changes daily if desired.

what it offers

  • Identify pluripotent stem cell colonies
  • Monitor pluripotency at different stages without compromising cell integrity
  • Preserve cell viability
  • Further propagate identified colonies

how it works

Pluripotent stem cell colonies can be easily identified in a few quick, simple steps using Alkaline Phosphatase Live Stain. This nonterminal assay can be used as often as desired to monitor changes in pluripotency without affecting cell viability. The stain is a soluble phosphorylated molecule that stains pluripotent colonies fluorescent green. Cells can then be easily observed using a FITC filter on a conventional microscope, while feeders and nonreprogrammed somatic cells remain unstained.


Alkaline Phosphatase Live Stain  
Alkaline Phosphatase Live Stain specifically stains pluripotent stem cells.
Product Quantity Cat. No.
Alkaline Phosphatase Live Stain 50 µL A14353

Study Nucleostemin With a New Recombinant Antibody―ABfinity™ Recombinant Antibody

what they are
Nucleostemin, also known as guanine nucleotide-binding protein-like 3 (GNL3), is a stem cell marker found in the nucleoli of embryonic stem (ES) cells, CNS stem cells, primitive cells in bone marrow, and cancer cells. This marker is known to interact with the tumor suppressor protein p53. Nucleostemin plays a role in controlling cell proliferation rate and apoptosis level in ES cells and ES cell–derived neural stem/progenitor cells.

what they offer

  • Consistent results
  • 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. This production process offers consistent lot-to-lot antibody performance. ABfinity™ oligoclonal antibodies are a mixture of recombinant monoclonal antibodies. This combines the improved signal strength that can come from using a polyclonal antibody with the highly reproducible results you get from ABfinity™ monoclonal antibodies.


Stem Cell Marker Nucleostemin  
Immunocytochemistry analysis of HeLa cells stained with Nucleostemin ABfinity™ Recombinant Rabbit Oligoclonal Antibody. (A) Alexa Fluor® 488 goat anti–rabbit IgG was used as a secondary antibody (green). (B) DAPI was used to stain nuclei (blue). (C) Alexa Fluor® 594 phalloidin was used to stain actin (red). (D) Composite image of cells showing nuclear localization of nucleostemin.

New FP-Based Labels for Key Cellular Targets—CellLight® Reagents

what they are
CellLight® reagents are based on a modified baculovirus containing fluorescent protein (FP) fusion constructs. These reagents combine the utility of fluorescent protein labels with the transduction efficiency of BacMam technology, enabling unambiguous labeling of cellular structure and processes in live mammalian cells. We now offer a complete selection of CellLight® reagents for a variety of targets.

what they offer

  • High efficiency—greater than 90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
  • Speed and convenience—add one or more CellLight® reagents to your cells, incubate overnight, and image (or store frozen, assay-ready cells for later use); easily adjust expression by varying the dose if desired
  • Robustness—CellLight® reagents are nonreplicating in mammalian cells, lack observable cytopathic effects, and are classified for biosafety level (BSL) 1 handling

how they work

BacMam 2.0 incorporates elements that enhance transduction efficiency and expression levels compared to BacMam 1.0, through more efficient cell entry, a stronger promoter, and enhanced mRNA stability. Unlike plasmid-based methods, there is no need for harsh transfection methods or tedious cloning. To achieve highly efficient labeling even in sensitive models like stem cells, neurons, and primary cells, just add CellLight® reagents to cells in complete medium, incubate, and image the next day.


New CellLight® Reagents  

Simple, efficient gene expression with BacMam 2.0. Using BacMam 2.0 to express genes is as simple as it is efficient. Simply add the virus to cells in complete medium, then analyze transduction efficiency the next day or freeze for future use. Cells can be transduced in suspension by mixing with the BacMam virus immediately prior to plating. Alternatively, adherent cells can be transduced by adding the virus to cells already in a dish or plate.

Note that for some cell types (primary neurons, some stem cells) a briefer treatment is preferable. Transduced cells can be divided into aliquots and stored frozen.



Creating a Tumor-Homing Particle for Imaging Studies

Click chemistry is a rapid, specific, and efficient strategy for labeling biological samples. Relying on the copper-catalyzed formation of a triazole from an azide-containing moiety and an alkyne-containing moiety, click chemistry–labeled reagents are stable and the reaction can be used to label even small molecules such as nucleotides, sugars, and amino acids. A wide variety of Molecular Probes® azide- and alkyne-containing dyes, haptens, and biomolecules are available for use in click reactions.

Nanoparticles are attractive probes for molecular imaging because they can be attached to targeting molecules and other functional groups, helping to ensure specificity and a high affinity for the molecule under study. Viral nanoparticles (VNPs) are naturally occurring materials that have several advantages over synthetic versions, including biodegradability, safety (in terms of human health), and full characterization (structure is mapped to atomic resolution). Steinmetz and colleagues adapted one type of VNP—cowpea mosaic virus (CPMV)—to create a long-term intravital imaging tool for the study of metastatic prostate cancer. The team used click chemistry to attach bombesin (a molecule that binds to the cell-surface gastrin-releasing peptide (GRP) receptors that are overexpressed in a variety of human tumors) and the near-infrared dye Alexa Fluor® 647 to the surface of CPMV and assessed the tumor-homing efficiency of the resulting particle. They found that the bombesin-conjugated nanoparticles accumulated in human prostate cancer cells, and they were able to see and quantify the uptake of these particles in real time. These studies demonstrate that the bombesin-modified CPMV particle is a promising molecular imaging agent.

  1. Steinmetz DF, Ablack AL, Hickey JL et al. (2011) Intravital Imaging of Human Prostate Cancer Using Viral Nanoparticles Targeted to Gastrin-Releasing Peptide Receptors. Small 7:1664–1672.


Click Chemistry  
Click azide/alkyne reaction. The azide and alkyne moieties are interchangeable. The molecule can be labeled with an alkyne and reacted with a fluorophore or hapten-azide.


Azide- or alkyne-modified fluorophores and haptens.

Label Ex/Em* Azide or alkyne Cat. No. Use
Alexa Fluor® 488 495/519 Azide A10266 Fluorescent dye or hapten
Alkyne A10267
Alexa Fluor® 555 555/565 Azide A20012 Fluorescent dye
Alkyne A20013
Alexa Fluor® 594 590/617 Azide A10270
Alkyne A10275
Alexa Fluor® 647 650/655 Azide A10277
Alkyne A10278
Biotin Not applicable Azide B10184 Hapten
Alkyne B10185
Oregon Green® 488 496/524 Azide O10180 Fluorescent dye or hapten
Alkyne O10181
Tetramethylrhodamine 555/580 Azide T10182
Alkyne T10183
*Fluorescence excitation and emission maxima, in nm.



On the Web

Molecular Probes® Handbook


Navigating our Website

Having trouble finding The Molecular Probes® Handbook or SpectraViewer on the new site? Not to worry. Just go to the Molecular Probes® web page and find links to all of our great resources and products.

Imaging Corner

Rat granule neurons imaged on the FLoid™ Imaging Station
Click to enlarge

Neuronal Staining Captured Using the FLoid™ Cell Imaging Station

Rat granule neurons were transfected with untargeted Green Fluorescent Protein (GFP) and stained with Hoechst 33342 stain. Cells were fixed and imaged on the FLoid™ Cell Imaging Station.


From the Bench

Super-Resolution Imaging—A Search for the Ideal Fluorophore

Dempsey GT, Vaughan JC, Chen KH et al. (2011) Nat Methods 8:1027–1036.

Until recently, diffraction has limited the spatial resolution of light microscopy. One of the super-resolution fluorescence imaging approaches that overcomes these limitations—stochastic optical reconstruction microscopy (STORM)— involves sequential activation and localization of individual fluorophores to achieve high spatial resolution. In a recent publication, Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging, Dempsey and colleagues surveyed 26 organic dyes for their performance in STORM, emphasizing two properties that the ideal fluorophore should possess: (1) high photon yield per switching event, and (2) a low duty cycle (i.e., low fraction of time the fluorophore spends in the “on” state). This combination provides both high localization precision and high localization density, which is ideal for resolving small structures. The dyes evaluated in the study spanned the visible and near-infrared range and included Molecular Probes® Alexa Fluor® dyes, fluorescein, FITC, rhodamine dyes, Cy® dyes, Atto dyes, Dyomics 654, and DyLight® dyes. From among several dyes with good overall properties, Alexa Fluor® 647 dye emerged as a top choice, allowing visualization of the hollowness of immunostained microtubule filaments. Amine-reactive dyes were used in this study, but other reactive forms that enable conjugation of these fluorophores to sulfhydryls, or even the use of click chemistry, are also available.

Molecular Probes® Webinar Series

Molecular Probes Webinar Series


February Webinar: A Comparison of Basic Immunofluorescent Labeling Strategies


In this free, live webinar, we'll compare different immunofluorescent labeling strategies and explore the pros and cons of each method. You'll learn when the use of a direct conjugate is appropriate and when amplification techniques can be used. We’ll also present a simple decision tree to aid you in determining the best method for each situation.

A Comparison of Basic Immunofluorescent Labeling Strategies
Date: Thursday, February 16, 2012
Times: 7:00 a.m. PST, 4:00 p.m. PST 

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