In This Issue


ReadyProbes™ Nuclear Stains   Nuclear Staining Has Never Been Easier—ReadyProbes™ Nuclear Stains for Live and Fixed Cells
Qdot® Primary Antibody Conjugates for Flow Cytometry   New Qdot® Primary Antibody Conjugates for Flow Cytometry—New CD19 and CD45R Antibodies
ABfinity™ Recombinant Antibodies   ABfinity™ Recombinant Antibodies—New Antibodies for RPS6


—The EVOS® FL Auto Imaging System

Follow Cell Biology EffortlesslyFully Automated EVOS® FL Auto Imaging System



Nuclear Staining Has Never Been Easier—ReadyProbes™ Nuclear Stains for Live and Fixed Cells

What They Are
Cell-permeant and -impermeant nuclear stains in ReadyProbes™ format are available in multiple colors. All emit bright fluorescence when bound to DNA and are formulated in a room temperature–stable solution and come in convenient dropper bottles. Two drops per milliliter is all it takes to stain your cells.

What They Offer

  • Exceptionally bright fluorescence when bound to DNA
  • Rapid staining of live or dead/fixed cells without wash steps, using a convenient dropper bottle
  • Room temperature–stable formulation eliminates need to dilute, weigh, or pipet

How They Work
The cell-permeant NucBlue® and NucRed™ Live reagents stain DNA and nuclei of both live and fixed cells and can be used for counterstaining, DNA content, and total cell counting. The membrane-impermeant NucBlue®, NucGreen™, and NucRed™ Dead stains easily stain cells with compromised cell membranes, yet don’t enter living cells. They are ideal for discrimination of live and dead cells, and staining of fixed cells and tissue slices. Finally, propidium iodide is a classic dead-cell marker for flow cytometry applications and is here made available in ReadyProbes™ format.

ReadyProbes Nucelar Stains   Rapid staining of dead cells using NucGreen™ Dead stain. HeLa cells were treated with 0.5 µM staurosporine to induce apoptosis and cell death and stained with NucGreen™ Dead 488 ReadyProbes™ Reagent (2 drops of reagent per milliliter of medium). Overlaid DIC and green channel images were collected over a 6 hr period using a DeltaVision® Core microscope with a 40x objective. Images are gain- and exposure-matched.

Time course of cell death visualized using ReadyProbes™ reagents. 
A549 cells grown in a MatTek glass-bottom dish were stained with both NucRed™ Dead 647 ReadyProbes™ Reagent and NucGreen™ Dead 488 ReadyProbes™ Reagent by adding 2 drops of the reagents per milliliter of complete medium and incubating for 20 min prior to disrupting membrane integrity by addition of DMSO/Triton® X-100 surfactant. The time-lapse video was acquired using a 40x objective on a DeltaVision® Core microscope using DIC, green, and far-red filter channels to show green dead nuclear staining in damaged cells.

New Qdot® Primary Antibody Conjugates for Flow Cytometry—New CD19 and CD45R Antibodies

What They Are
Qdot® primary conjugates for the CD19 mouse anti-human marker and CD45R (B220) rat anti-mouse marker are now available in new colors for flow cytometry.

What They Offer

  • Compatibility—combine with existing organic dyes to increase the number of detectable parameters
  • Stability—do not degrade over time like tandem conjugates, affording greater reproducibility
  • Minimal single-laser compensation—narrow emission spectra allow for minimal compensation when using a single excitation source

How They Work
Qdot® primary conjugates have extremely bright fluorescence emission that makes them well suited for the detection of low-abundance extracellular proteins. Efficient optical excitation is possible using the 405 nm violet excitation light source. In addition, the narrow, symmetrical emission profiles of Qdot® nanocrystal conjugates require substantially lower compensation, enabling better, more efficient multicolor assays using the violet laser.

Histogram overlay of live mouse splenocytes

Histogram overlay of live mouse splenocytes. The black line represents cells labeled with CD45R (B220) rat anti-mouse Qdot® 800 conjugate, and the gray line represents unstained cells. Samples were acquired and analyzed using 405 nm excitation and a 780/60 bandpass emission filter on the Attune® Acoustic Focusing Cytometer with Blue/Violet option.

ABfinity™ Recombinant Antibodies—New Antibodies for RPS6

What They Are
ABfinity™ recombinant monoclonal and oligoclonal antibodies offer consistent results, minimizing the need to revalidate working antibody dilutions for your experiments each time you order. Over the past year we have launched several new ABfinity™ recombinant antibodies.

Ribosomal protein 6 (RPS6) is a component of the 40S ribosomal subunit belonging to the S6E family of ribosomal proteins. RPS6 is found in the cytoplasm, and it mediates translation initiation at the 5´-m7GpppG cap of mRNA. It is a key substrate for protein kinases, and it is phosphorylated by growth factors and mitogens during cell growth and cell division.

What They Offer

  • Specificity—undergo rigorous validation
  • High performance—proven consistency from lot to lot
  • Efficiency—detect low-level targets, with less sample

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, with the highly reproducible results you get from ABfinity™ monoclonal antibodies.

Hela cells labeled with ABfinity™ RPS6 Recombinant Rabbit Monoclonal Antibody  
Flow cytometry analysis of HeLa cells labeled with ABfinity™ RPS6 Recombinant Rabbit Monoclonal Antibody. Fixed and permeabilized HeLa cells were labeled with ABfinity™ RPS6 recombinant rabbit monoclonal antibody, followed by Alexa Fluor® 488 goat anti–rabbit IgG staining (right peak, filled). To confirm specificity, the cells were labeled with an isotype control and stained using the same Alexa Fluor® 488 secondary antibody.



Follow Cell Biology Effortlessly—Fully Automated EVOS® FL Auto Imaging System

The EVOS® FL Auto Imaging System makes it easy to capture high-quality fluorescence images, including automated time-lapse imagery. To demonstrate the capabilities of this system, U2OS cells were grown overnight in McCoy’s Medium + 10% FBS. For imaging, McCoy’s Medium was replaced with Live Cell Imaging Solution (LCIS). To identify living and dead cells, components of the LIVE/DEAD® Cell Imaging Kit 488/570 were added to the cells in LCIS. The LIVE/DEAD® Cell Imaging Kit distinguishes live cells by the presence of ubiquitous intracellular esterase activity, as determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant calcein AM to the intensely fluorescent calcein (green), which is well retained within live cells. The red component of the LIVE/DEAD® Cell Imaging Kit is cell-impermeant and therefore only enters cells with damaged membranes. In dying and dead cells, bright red fluorescence is generated upon binding to DNA. In this video, this transformation can be seen following addition of 1 µM staurosporine to induce cell death. Cells were imaged on the new EVOS® FL Auto Imaging System with time-lapse images taken every 5 minutes for a period of 14 hours.

Time course of cell death visualized using the LIVE/DEAD® Cell Imaging Kit. The LIVE/DEAD® Cell Imaging Kit is based on a cell-permeant dye (calcein AM) that stains live cells bright green, and a cell-impermeant red marker that only stains dead and dying cells, which are characterized by compromised cell membranes. Labeled U2OS cells were treated with 1 µM staurosporine, and fluorescence images in the FITC and Texas Red® channels were acquired every 5 minutes over 14 hours on the EVOS® Auto Imaging System using a 20x objective.


Detecting Superoxide Formation in Mitochondria With MitoSOX™ Red Superoxide Indicator

MitoSOX™ Red mitochondrial superoxide indicator—comprising hydroethidine (HE) covalently linked to a triphosphonium cation through a hexyl carbon chain—permeates live cells, where the positive charge selectively targets the mitochondria and accumulates as a function of mitochondrial membrane potential. Rapidly oxidized by superoxide but not by other reactive oxygen species (ROS) and reactive nitrogen species (RNS), the MitoSOX™ Red reagent exhibits bright red fluorescence upon oxidation and subsequent binding to mitochondrial nucleic acids. The MitoSOX™ Red probe is typically measured using 510 nm excitation; however, to more selectively detect superoxide, it can be excited at 396 nm to diminish autooxidation of ethidium.


MitoSOX™ Red Superoxide Indicator
Visualizing glucose-mediated oxidative stress. Live human osteosarcoma (U2OS) cells were plated in Minimum Essential Medium (MEM) and incubated overnight at 37°C with CO2. To mitigate the effect of high glucose–mediated oxidative stress, samples B–D were washed in PBS and immersed in low-glucose Dulbecco’s Modified Eagle Medium (DMEM) and incubated overnight at 37°C with CO2. Samples were then washed in Hanks' Balanced Salt Solution (HBSS) and then treated as follows for the next 30 minutes: (A, B) cells were left in HBSS; (C) cells were incubated in HBSS + 100 μM antimycin A; (D) cells were incubated in HBSS + 100 μM DEANO. MitoSOX™ Red reagent at 5 µM was added to each sample, and cells were incubated for 30 minutes and imaged by confocal microscopy.



On the Web

Fluorescence Spectraviewer


The SpectraViewer Tool Takes a Broader View


We are very excited to announce the release of the new and improved SpectraViewer tool. The table below highlights the new and updated features, and the new tool—unlike the original version that only offered spectral data for Molecular Probes® fluorophores—will continually be updated to include as many spectra, instruments, and filters as possible.

  Original SpectraViewer 2013 SpectraViewer
Number of fluorophores you can plot on one graph 4 14
Number of lasers you can designate per graph 1 7
Number of excitation filters allowed per graph 3 ≥10
Number of emission filters allowed per graph 5 ≥10
Load standard instrument configurations with one click No Yes
Print or save SpectraViewer graph No Yes
View available products No Yes
Select fluorophores based on application No Yes
Save frequently used SpectraViewer settings No Yes
Download spectra files from SpectraViewer No Yes
Normalize spectra to laser excitation source No Yes
View calculated spectra emission spillover into emission filters No Yes

Imaging Corner

Touch Screen Controls Give Image Zoom Without Resolution Loss

The versatility and ease of use of the EVOS® FL Auto Imaging System was demonstrated in this imaging experiment. A stained cross-section of rat ileum was scanned and stitched on the EVOS® FL Auto Imaging System using a 10x objective with color camera accessory. Total acquisition and processing time was 8 minutes. The file was exported as a .jpg file, and was viewed in a “pinch-and-zoom” format on touch screen devices without loss of resolution. In addition, users can choose to scan and stitch an image at any magnification. The resolution of the image in the pinch-and-zoom format automatically increases with increasing objective power. The same tissue was imaged at 2x, 4x, 10x, 20x, and 40x on the EVOS® FL Auto Imaging System.

From the Bench

FUEL Enhances Bioluminescence Signals for a Range of Applications

Dragavon J, Blazquez S, Rekiki A et al. (2012) Proc Natl Acad Sci USA 109:8890–8895.
doi: 10.1073/pnas.1204516109.

There is great potential for imaging applications based on bioluminescent reporters, but the blue light (~490 nm) of various lux reporter systems is readily absorbed in mammalian cells and tissues, making its use limited in these types of investigations. In a recent publication, Dragavon and colleagues report a new method to overcome these limitations. Termed FUEL (fluorescence by unbound excitation from luminescence), this approach involves pairing blue light–emitting bacteria with Qtracker® 705 particles to enhance the detectable light output from the bioluminescence source. In fact, they were able to observe signals up to an order of magnitude greater than without enhancement. The authors postulate that FUEL could be useful in both in vitro and in vivo imaging applications.

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