In This Issue

FEATURED NEW PRODUCTS

Qdot® Nanocrystals Exceptionally Bright and Photostable—Donkey Secondary Antibodies Conjugated to Qdot® Nanocrystals
   
Adiponectin‑ABfinity™ Study the Role of Adiponectin in Metabolism—New ABfinity™ Recombinant Antibodies
   
Premo™ FUCCI Analyze Cell Cycle in Any Live-Cell Model—Premo™ FUCCI Cell Cycle Sensor
   
BackDrop™ Background Suppressor Cut Through the Haze—BackDrop™ Background Suppressor
   
New Antibodies See all of this month's New Antibodies

NEW APPLICATIONS

Fluorescence LabelingVisualize Virus–Cell Interactions Using a Simple Fluorescence Labeling Method

PROVEN PERFORMERS

Tali™ Image-Based CytometerMeasuring GFP and RFP Expression Using the Tali™ Image-Based Cytometer

OTHER PUBLICATIONS

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

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FEATURED NEW PRODUCTS

Exceptionally Bright and Photostable―Donkey Secondary Antibodies Conjugated to Qdot® Nanocrystals

what they are
Qdot® nanocrystal–conjugated antibodies are exceptionally bright and photostable. We now offer 18 new donkey secondary antibodies conjugated to Qdot® nanocrystals. Donkey anti-mouse, donkey anti-rabbit, and donkey anti-goat conjugates are provided in six of the most commonly used colors: Qdot® 525, Qdot® 565, Qdot® 585, Qdot® 605, Qdot® 625, and Qdot® 655. In addition, we offer goat anti-mouse, goat anti-rabbit, goat anti-human, goat anti-rat, and rabbit anti-goat Qdot® nanocrystal secondary antibodies.



what they offer

  • Brightness—as much as 50 times brighter than organic dyes
  • Photostability—no photobleaching, unlike organic dyes
  • Multiplexing capability—six colors, from Qdot® 525 to Qdot® 655


how they work

Qdot® nanocrystals are composed of semiconductor material, generating a photostable fluorescent particle. Unlike even the best organic fluorescent dyes, Qdot® nanocrystals do not photobleach over time.

 

Qdot™ nanocrystal-labeled secondary antibodies 
HeLa cells stained with Qdot® nanocrystal–labeled secondary antibodies. Cells were incubated with mouse anti–β-tubulin and then incubated with Qdot® 605 Donkey Anti–Mouse IgG Conjugate (green). Nuclei were stained with Qnuclear™ Deep Red Stain (red).
Product Quantity Cat. No.
Qdot® 525 Donkey Anti–Goat IgG Conjugate (H+L), 1 µM solution100 µLQ22072
Qdot® 525 Donkey Anti–Mouse IgG Conjugate (H+L), 1 µM solution100 µLQ22073
Qdot® 525 Donkey Anti–Rabbit IgG Conjugate (H+L), 1 µM solution100 µLQ22074
Qdot® 565 Donkey Anti–Goat IgG Conjugate (H+L), 1 µM solution100 µLQ22075
Qdot® 565 Donkey Anti–Mouse IgG Conjugate (H+L), 1 µM solution100 µLQ22076
Qdot® 565 Donkey Anti–Rabbit IgG Conjugate (H+L), 1 µM solution100 µLQ22077
Qdot® 585 Donkey Anti–Goat IgG Conjugate (H+L), 1 µM solution100 µLQ22078
Qdot® 585 Donkey Anti–Mouse IgG Conjugate (H+L), 1 µM solution100 µLQ22079
Qdot® 585 Donkey Anti–Rabbit IgG Conjugate (H+L), 1 µM solution100 µLQ22080
Qdot® 605 Donkey Anti–Goat IgG Conjugate (H+L), 1 µM solution100 µLQ22081
Qdot® 605 Donkey Anti–Mouse IgG Conjugate (H+L), 1 µM solution100 µLQ22082
Qdot® 605 Donkey Anti–Rabbit IgG Conjugate (H+L), 1 µM solution100 µLQ22083
Qdot® 625 Donkey Anti–Goat IgG Conjugate (H+L), 1 µM solution100 µLQ22084
Qdot® 625 Donkey Anti–Mouse IgG Conjugate (H+L), 1 µM solution100 µLQ22085
Qdot® 625 Donkey Anti–Rabbit IgG Conjugate (H+L), 1 µM solution100 µLQ22086
Qdot® 655 Donkey Anti–Goat IgG Conjugate (H+L), Qdot® VIVID, 1 µM solution100 µLQ22087
Qdot® 655 Donkey Anti–Mouse IgG Conjugate (H+L), Qdot® VIVID, 1 µM solution100 µLQ22088
Qdot® 655 Donkey Anti–Rabbit IgG Conjugate (H+L), Qdot® VIVID, 1 µM solution100 µLQ22089

Study the Role of Adiponectin in Metabolism—New ABfinity™ Recombinant Antibodies

what they are
Adiponectin is a hormone exclusively produced in adipose tissue and secreted into plasma. Adiponectin is involved in a number of metabolic processes, including glucose regulation and fat metabolism. ABfinity™ adiponectin recombinant monoclonal and oligoclonal antibodies offer consistent results, minimizing the need to revalidate working antibody dilutions each time you order.



what they offer

  • Consistent results
  • Minimized need to revalidate working antibody dilutions each time you order
  • New ABfinity™ antibodies released every month


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. Our Adiponectin ABfinity™ Recombinant Rabbit Antibodies are validated for western blotting, immunocytochemistry, and indirect ELISA applications.

 

Immunocytochemistry analysis of HeLa cells 

 

Immunocytochemistry analysis of HeLa cells. Cells were treated with Adiponectin ABfinity™ Recombinant Rabbit Monoclonal Antibody and labeled with Alexa Fluor® 488 goat anti-rabbit secondary antibody (green). Actin was stained with Alexa Fluor® 594 phalloidin (red), and nuclei were stained with DAPI (blue). The image shows subcellular localization in the perinuclear region.

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

what it is
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 Premo™ FUCCI Cell Cycle Sensor to your cells in complete medium, incubate overnight, and analyze


how it works

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 Premo™ FUCCI Cell Cycle Sensor. U2OS cells were transduced with 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.

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.

 

NEW APPLICATIONS

Visualize Virus–Cell Interactions Using a Simple Fluorescence Labeling Method

  • Maintain virus function using simple new labeling method
  • Alexa Fluor® dyes offer bright, photostable, pH-independent fluorescence across the spectrum
  • pHrodo™ red dye offers real-time monitoring of phagocytosis and endocytosis


Researchers recently developed a new method for directly labeling viruses with fluorescent dyes. The team used the amine-reactive succinimidyl ester (SE) form of Alexa Fluor® dyes. Rather than DMSO as the recommended solvent, the water-soluble Alexa Fluor® dyes were dissolved in sodium bicarbonate buffer and added directly to the virus (J Virol Methods 167:172 (2010)). Using this method, attachment of the Alexa Fluor® label did not affect the ability of the virus to bind and enter the host cell.

This protocol is also available as a video.

The pHrodo™ Red succinimidyl ester is another reagent that can potentially be used in this protocol. Unlike the Alexa Fluor® dyes, pHrodo™ Red SE exhibits pH-sensitive red fluorescence that increases in intensity with increasing acidity. pHrodo™ dye is essentially dark in the extracellular environment but exhibits bright red fluorescence at pH 5–7 as it undergoes endocytosis, enabling real-time monitoring without requiring any extra wash steps or dye quenchers.

 

Visualizing internalization

Visualizing virus internalization. CellLight® MAP4-GFP, a BacMam virus, was labeled in serum-free buffer with pHrodo™ SE. U2OS cells were placed on a microscope stage inside a climate control chamber, and 50 µL of labeled virus (100 particles/cell) was added. A fluorescent image acquired with the appropriate filters (TRITC) for pHrodo™ dye is shown in a differential interference contrast overlay (A). Red fluorescence from the labeled viruses can be seen within intracellular vesicles. Cells were returned to the incubator overnight and imaged again the following day. Fluorescence from CellLight® MAP4-GFP (microtubules) can be seen, as can residual pHrodo™ staining from viral remnants (B), showing that labeled viruses retain functionality following pHrodo™ SE labeling.

PROVEN PERFORMERS

Measuring GFP and RFP Expression Using the Tali™ Image-Based Cytometer

Accurately quantifying fluorescent protein expression can be challenging. The Tali™ Image-Based Cytometer measures cellular fluorescence that falls within the green (Ex/Em = 458/525 nm) or red (Ex/Em = 530/585 nm) channels, which allows researchers to identify and quantify populations of cells that are differentially expressing Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP). Results generated by the Tali™ instrument are comparable to results from a flow cytometer.

Four mammalian cell lines were transduced with CellLight® Nucleus-GFP and CellLight® Plasma Membrane-RFP. Using the bright-field and fluorescence channels of the Tali™ instrument, cell counts and fluorescence data were generated and compared to results from a flow cytometer. The percentages of cells expressing each reporter protein as recorded by the Tali™ instrument and a flow cytometer are shown in the table. The transduction efficiency for each of the four cell types was at least 35%, and population statistics for each combination of fluorescent proteins matched well between the Tali™ Image-Based Cytometer and a flow cytometer.

 

Fluorescent protein expression data (percentage of cells expressing fluorescent reporter protein) from the Tali™ Image-Based Cytometer are comparable to those obtained using flow cytometry.

Cell Type
HEKn
293MSR
U2OS
CHO-S
 Tali™ cytometerFlow cytometerTali™ cytometerFlow cytometerTali™ cytometerFlow cytometerTali™ cytometerFlow cytometer
GFP+/RFP22%26%4%0%2%4%13%7%
GFP/RFP+11%12% 1%2%4%2%6%7%
GFP+/RFP+54%49%44%40%32%35%36%27%

 

GFP and RFP expression 
Population analysis of GFP- and RFP-expressing cells. The GFP+RFP display on the Tali™ Image-Based Cytometer is shown after measuring U2OS cells transduced with CellLight® Nucleus-GFP and CellLight® Plasma Membrane-RFP. A representative image of cell fluorescence is shown on the left side of the screen, while quantitative population data (% GFP positive, % RFP positive, % GFP and RFP positive, and % GFP and RFP negative) are shown on the right side of the screen.
Product Quantity Cat. No.
Tali™ Image-Based Cytometer1 instrumentT10796

 

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Imaging Corner

Enlarge Image  

Mitochondrial and Cytoskeletal Staining in Endothelial Cells

Mitochondria in bovine pulmonary artery endothelial cells were labeled with MitoTracker® Red CMXRos (pseudocolored yellow). Cells were then fixed and permeabilized. F-actin was stained with Alexa Fluor® 488 phalloidin (green), and nuclei were stained with DAPI (pseudocolored red). Imaging was performed using a Zeiss LSM 710 confocal microscope. Image contributed by Nicholas Dolman, Life Technologies Corporation.

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