ProbesOnline™ Special Edition: The Most Popular Articles of 2012

BackDrop™ Background Suppressor Cut Through the Haze—BackDrop® Background Suppressor
   
NucBlue™ The Clear Imaging Choice—Live Cell Imaging Solution and NucBlue® Live and Fixed Cell Stains
   
Image-iT® Fixation/Permeabilization Kit A Fix for Your Immunofluorescence Samples—Image-iT® Fixation/Permeabilization Kit
   
LIVE/DEAD® Cell Imaging Kit Imaging Dead and Alive—LIVE/DEAD® Cell Imaging Kit
   
CellLight® Actin-RFP BacMam 2.0 Visualize Actin Before Fixing Your Cells—CellLight® Actin-RFP BacMam 2.0
   
CellROX® Fluorescent Reagents Oxidative Stress Detection—CellROX® Fluorescent Reagents
   
pHrodo™ Bioparticles® Conjugates 

Visualize Phagocytosis, Endocytosis, and Internalization—pHrodo™ Red and Green Reactive Dyes and pHrodo™ BioParticles® Conjugates

   
Introducing the Attune® Autosampler Your Flow Experiments Just Got Faster and Easier—Introducing the Attune® Autosampler
   
New Alexa Fluor® 790 Secondary Antibodies Fluorescent Western Blotting—New Alexa Fluor® 680 and 790 Secondary Antibodies
   
Premo™ FUCCI Analyze Cell Cycle in Any Live-Cell Model—Premo™ FUCCI Cell Cycle Sensor

 

 

FROM THE BENCH

Visualizing Cancer Cells With the FLoid® Cell Imaging Station
Researchers used the FLoid® instrument in cancer cell migration and proliferation studies.

 UPCOMING MEETINGS

American Society for Cell Biology (ASCB)
December 15–19
San Francisco, CA
Booth #415

OTHER PUBLICATIONS

BioProbes® Journal for Cell Biology Applications
BioProbes 68 
 
The Molecular Probes® Handbook
Molecular Probes Handbook
 

 

Cut Through the Haze—BackDrop™ Background Suppressor

What It Is
BackDrop® Background Suppressor is a set of novel Molecular Probes® reagents designed to effectively suppress background fluorescence during live-cell imaging. If you are experiencing high background signal or weak fluorescence in the blue, green, or red channels, see how BackDrop® Background Suppressor improves your results by cutting through the haze and increasing contrast.



What It Offers

  • Greatly reduced background fluorescence in live-cell imaging with dyes and fluorescent proteins
  • Higher signal-to-noise ratio and improved sensitivity for better results
  • Convenience of direct addition to cells from dropper bottle


How It Works

Background fluorescence from media components and extracellular dyes decreases signal-to-noise ratio and assay sensitivity. BackDrop® Background Suppressor is a direct-addition product provided as a 6-pack of ultraconvenient dropper bottles, with two vials each of blue, green, and red background suppressors. Apply two drops per milliliter of your live-cell sample to help eliminate background haze, improve image quality, and help eliminate the need for medium removal with its risk of potential cell loss.

 

BackDrop™ Background Suppressor 
Effect of BackDrop® Background Suppressor. Bovine pulmonary artery endothelial cells in DMEM + 20% FBS, imaged without (–) and with (+) BackDrop® Background Suppressor.

 



The Clear Imaging Choice—Live Cell Imaging Solution and NucBlue® Live and Fixed Cell Stains

What They Are
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.

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—provided in convenient dropper bottles. Just use two drops per milliliter to stain your cells.



What They Offer

  • Physiological and chemical optimization—Live Cell Imaging Solution affords more efficient imaging and minimal background
  • Extended buffering capacity compared to other media―Live Cell Imaging Solution allows improved cell viability
  • Convenience―ready-to-use liquid formulations of room temperature–stabilized, high-purity Hoechst 33342 and DAPI in dropper bottles


How They Work

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.

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 convenient 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 stained with NucBlue® Fixed Cell Stain, BODIPY® FL phallacidin, and MitoTracker™ Red CMXRos and imaged on the FLoid® Cell Imaging Station.



A Fix for Your Immunofluorescence Samples—Image-iT® Fixation/Permeabilization Kit

What It Is
The Image-iT® Fixation/Permeabilization Kit contains all of the necessary reagents to prepare your cells for antibody staining and imaging. The high-quality components help preserve cell morphology and reduce background staining, and are provided in a convenient storage box in single-use vials with easy-to-follow protocols.



What
It Offers

  • A complete fixation/permeabilization kit with ready-to-use components and room temperature storage
  • Optimized for secondary antibody labeling, fixed-cell dye staining, and applications involving Green Fluorescent Protein (GFP)
  • Compatible with analysis of most cellular antigens


How
It Works
Cells are treated sequentially with the fixative, the permeabilization solution, and the blocking buffer according to a simplified and easy-to-follow protocol. The reagents are optimized to preserve 3D cellular structure and provide adequate antibody access to intracellular antigenic sites. The high-quality components and optimized formulations help decrease background staining, improving signal-to-noise levels and image quality. In extensive testing with both nuclear and cytoplasmic targets, the reagents provided in this kit offered results equal or superior to those from standard reagents.

 

Image-iT® Fixation/Permeabilization Kit
Fluorescently labeled cells fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit. (A)
Neonatal human dermal fibroblasts were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit. Peroxisomes were detected with an anti-PMP70 antibody and labeled with
Alexa Fluor® 488 goat anti–rabbit IgG. Actin was labeled with Alexa Fluor® 594 phalloidin, and the nucleus was stained with Hoechst 33342. Images were acquired using a Nikon® upright microscope (60x). (B) Bovine pulmonary artery endothelial cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit. Mitochondria were detected with ATP Synthase Subunit IF1 Monoclonal Antibody and labeled with Alexa Fluor® 488 goat anti–mouse IgG. Actin was labeled using Alexa Fluor® 594 phalloidin, and the nucleus was stained with Hoechst 33342. Images were acquired on a Nikon® upright microscope (60x).
Product Quantity Cat. No.
Image-iT® Fixation/Permeabilization Kit1 kitR37602



Imaging Dead and Alive—LIVE/DEAD® Cell Imaging Kit

What It Is
The new LIVE/DEAD® Cell Imaging Kit is a sensitive, two-color fluorescence cell viability assay optimized for FITC and Texas Red® filters. Quick and easy to use, the kit provides simultaneous determination of live and dead cells with two probes that measure recognized parameters of cytotoxicity and cell viability—intracellular esterase activity and plasma membrane integrity.



What
It Offers

  • Simultaneous detection of live and dead cells
  • Convenient, fast, and accurate detection
  • Sensitive probes ideal for FITC and Texas Red® filters


How
It Works
The LIVE/DEAD® Cell Imaging Kit includes a cell-permeant component for staining live cells and a cell-impermeant component for staining dead and dying cells, which are characterized by compromised cell membranes. The LIVE/DEAD® Cell Imaging Kit components are optimized for common imaging filters (FITC and Texas Red®). The live-cell dye produces an intense, uniform green fluorescence in live cells (excitation/emission maxima ~495/515 nm). The red dead-cell dye (excitation/emission maxima ~570/602 nm) predominantly stains nuclei in cells with compromised cell membranes, which are a strong indicator of cell death and cytotoxicity.

 

LIVE/DEAD® Cell Imaging Kit 
Fluorescence staining of live and dead cells. HepG2 cells grown in 96-well microplates were stained with the LIVE/DEAD® Cell Imaging Kit and imaged with a Nikon Eclipse® T200 microscope. Live cells are stained green, and dead cells are stained red.
Product Quantity Cat. No.
LIVE/DEAD® Cell Imaging Kit, 488/5701 kitR37601



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

What It Is
The 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—cotransduce more than one BacMam reagent for multiplex experiments or colocalization 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 cotransduced with CellLight® Actin-RFP and CellLight® Talin-GFP and imaged on a Zeiss® LSM confocal microscope after overnight incubation.



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, measured with CellROX® Orange Reagent.
The cells 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)644/665545/565485/520
Live-Cell–CompatibleYesYesYes
Formaldehyde-FixableYesNoYes
Detergent-ResistantNoNoYes
Platforms*FC, HCS, HTS, I, Attune® systemFC, HCS, I, Tali® systemFC, HCS, HTS, I, and Tali®, FLoid®, and Attune® systems
Quantity5 x 50 µL5 x 50 µL5 x 50 µL1 kit
Cat. No.C10422C10443C10444C10448
* FC = flow cytometry; HCS = high-content screening; HTS = high-throughput screening; I = imaging.



Visualize Phagocytosis, Endocytosis, and Internalization—pHrodo™ Red and Green Reactive Dyes and pHrodo™ BioParticles® Conjugates

What They Are
The proprietary pH-sensitive pHrodo™ conjugates of E. coli, S. aureus, and zymosan are designed to be specific sensors of phagocytosis. These red- and green-fluorescent pHrodo™ BioParticles® conjugates can be visualized using standard TRITC or fluorescein (FITC) filters, respectively.

pHrodo™ STP esters and maleimides are reactive versions of our unique fluorogenic pH indicators, developed to allow researchers to label proteins or amine- or thiol-containing biomolecules. pHrodo™ Green indicators expand the multiplexing capabilities of this technology for the study of many membrane internalization processes.



What They Offer

  • Flexibility—label any amine- or thiol-containing molecule with a pH indicator dye
  • Multiplexing capability—available in red- and green-fluorescent versions
  • Utility—optimized, fixable probes for detecting phagocytosis in live cells
  • Compatibility—conjugates can be used for live-cell fluorescence imaging, high-content screening (HCS), flow cytometry, and high-throughput screening (HTS), and with benchtop instruments such as the Tali®, FLoid®, and Attune® systems


How They Work

During phagocytosis, cells ingest the pHrodo™ dye–conjugated BioParticles® and form phagosomes. These phagosomes fuse with early lysosomes to form acidic phagolysosomes. The nonfluorescent pHrodo™ dye, conjugated to the surface of the BioParticles® reagents, becomes fluorescent with this reduction in pH, making these particles ideal reagents for the study of phagocytosis and its regulation by drugs and environmental factors. Because pHrodo™ dye is minimally fluorescent at neutral pH, wash steps and quencher dyes are not required. To facilitate multiplexing, cells assayed for phagocytic activity with pHrodo™ BioParticles® conjugates can be fixed, preserving the fluorescent signal potentially for up to 24 hours.

The pHrodo™ green and red maleimides and amine-reactive esters can be used in a simple protocol for labeling reactions. The dyes are provided as dry powders that are reconstituted in DMSO to make the stock solutions. Protein to be labeled with an amine-reactive form of pHrodo™ dye should be in 0.1 M sodium bicarbonate buffer, pH 8.3, at a concentration of ≥1 mg/mL. The reactive dye is added to the protein solution and reacted for up to an hour. The resulting conjugate can be purified using standard gel filtration techniques. Thiols can be labeled in a similar manner; however, a protein reduction step may be required prior to the labeling reaction (see product manual).

 

Phagocytosis in MMM cells Phagocytosis in MMM cells visualized using pHrodo™ Green E. coli BioParticles® Conjugate and pHrodo™ Red E. coli BioParticles® Conjugate. MMM cells were plated in complete medium and cultured overnight. Cells were then rinsed with 1X Live Cell Imaging Solution and treated with a suspension of pHrodo™ Red E. coli BioParticles® Conjugate (left) and pHrodo™ Green E. coli BioParticles® Conjugate (right) in Live Cell Imaging Solution at 1 mg/mL. Cells were incubated for 90 min at 37°C. Nuclei were stained with NucBlue® Live Cell Stain.

 



Your Flow Experiments Just Got Faster and Easier—Introducing the Attune® Autosampler

What It Is
The Attune® Autosampler, an optional accessory to the Attune® Acoustic Focusing Cytometer, enables rapid processing of multiple samples. In addition, the newly improved software offers greater functionality for data analysis.



What It Offers

  • Broad compatibility—the Attune® Autosampler is compatible with many different formats, including tubes and 96-well, 384-well, and deep-well plates
  • Sample integrity and data quality—the Attune® Autosampler mixes sample by aspiration (instead of shaking) to help ensure sample uniformity
  • Easy operation—the Attune® Autosampler can be easily attached to and detached from the instrument, allowing users to quickly switch between the autosampler and individual tubes


How It Works

The Attune® Acoustic Focusing Cytometer uses proprietary sound wave technology to help ensure accurate alignment of cells through the path of the laser beam. The Attune® Autosampler enables high-throughput sample processing. Its intelligent probe leads to minimal clogging and carryover and also prevents instrument damage. Using the virtual plate layout view, users can create compensation wells, define instrument settings, and identify samples. The free software allows multiple experiments to be defined on a single plate.

 

The Attune® Acoustic Focusing Cytometer and Attune® Autosampler 
The Attune® Acoustic Focusing Cytometer and Attune® Autosampler.



Fluorescent Western Blotting―Alexa Fluor® 680 and Alexa Fluor® 790 Secondary Antibodies

What They Are
Alexa Fluor® 680 and Alexa Fluor® 790 secondary antibodies and streptavidin conjugates are ideal for fast and accurate multicolor western detection. Alexa Fluor® 680 and 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.

 

Simultaneous detection of AKT and GAPDH  Simultaneous detection of AKT and GAPDH. AKT protein (pseudocolored green) can easily be detected on the same blot as the housekeeping protein GAPDH (red). The ability to simultaneously detect proteins of two different sizes on the same blot allows simple normalization using a housekeeping protein as the standard. Western blots were probed with GAPDH Mouse mAb (Cat. No. 398600) and AKT (pan) Rabbit pAb (Cat. No. 44609G), followed by detection with Alexa Fluor® 680 goat anti–mouse IgG and Alexa Fluor® 790 goat anti–rabbit IgG antibodies. Lane 1: MagicMark™ XP Western Protein Standard (Cat. No. LC5602). Lanes 2–5: Serial dilutions of Jurkat cell lysate containing AKT.
Product Alexa Fluor® 680 Alexa Fluor® 790
StreptavidinS32358S11378
Goat anti–mouse IgGA21057A11375
Goat anti–mouse IgG (H+L), highly cross-adsorbedA21058A11357
Goat anti–rabbit IgG (H+L)A21076A11367
Goat anti–rabbit IgG (H+L), highly cross-adsorbedA21109A11369
Goat anti–rat IgG (H+L)A21096
Donkey anti–mouse IgG (H+L)A10038
Donkey anti–rabbit IgG (H+L)A10043A11371
Donkey anti–goat IgG (H+L)A21084A11370
Rabbit anti–mouse IgG (H+L)A21065
Rabbit anti–goat IgG (H+L)A21088



Analyze Cell Cycle in Any Live-Cell Model—Premo™ FUCCI Cell Cycle Sensor

What It Is
The Premo™ FUCCI Cell Cycle Sensor is a fluorescent two-color sensor of cell cycle progression and division in live cells. This reagent allows accurate and sensitive cell cycle analysis of individual cells or a population of cells by fluorescence microscopy, flow cytometry, or high-content imaging.



What It Offers

  • Accuracy―cell cycle–controlled expression of bright GFP and RFP indicators for live-cell analysis of individual cells or populations
  • High efficiency―>90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
  • Speed and convenience―simply add the Premo™ FUCCI Cell Cycle Sensor to your cells in complete medium, incubate overnight, and analyze


How It Works

The Premo™ FUCCI Cell Cycle Sensor is delivered by highly efficient BacMam 2.0 technology, enabling cell cycle studies in essentially any cell type. Provided in a ready-to-use format—simply add, incubate, and image—the Premo™ FUCCI sensor affords highly efficient transient expression in cell lines, primary cells, and stem cells. Cells change from red in the G1 phase to yellow in the G1/S interphase and green in the S, G2, and M phases, as fusions of emGFP and TagRFP coupled to two cell cycle–regulated proteins are expressed and degraded.

 

Premo™ FUCCI Cell Cycle SensorImaging cell cycle progression in live cells with the Premo™ FUCCI Cell Cycle Sensor. U2OS cells were transduced with the Premo™ FUCCI Cell Cycle Sensor. Images were acquired every 10 min for 16 hr. Imaging was performed on live cells using standard FITC/TRITC/Cy®5 filter sets.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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