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In This Issue

FEATURED NEW PRODUCTS

CellLight® Reagents Light. Cells. Action.—New CellLight® Reagents for Live-Cell Imaging
   
Premo™ Autophagy TR-FRET LC3B Quantify Autophagy—New Premo™ Autophagy TR-FRET LC3B Kits
   
CREB antibodies Study CREB and Downstream Signaling Partners—New Antibodies for CREB and p38 MAPK
   
New Antibodies See all of this month's New Antibodies

 NEW APPLICATIONS

ApoptosisTissue Homeostasis and Detection of Apoptosis

 PROVEN PERFORMERS

Click-iT® EdU Analysis of Cell Proliferation and Mitotic ArrestClick-iT® EdU Assays on the Tali™ Image-Based Cytometer

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BioProbes 66 

FEATURED NEW PRODUCTS

Light. Cells. Action.—New CellLight® Reagents for Live-Cell Imaging

what they are

CellLight® reagents combine the utility and selectivity of fluorescent proteins with the transduction efficiency of BacMam technology, enabling unambiguous staining of organelles, cellular structures, and processes in live mammalian cells. These reagents are provided in a ready-to-use format—simply add these reagents to your cell culture, incubate, and image. CellLight® reagents offer highly efficient expression in cultured cell lines, primary cells, stem cells, and neurons.

what they offer

  • Transduction efficiency—greater than 90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
  • Speed and convenience—simply add CellLight® reagents to your cells, incubate overnight, and image, or store frozen, assay-ready cells for later use
  • Safety for you and your cells—CellLight® reagents are nonreplicating in mammalian cells, lack observable cytopathic effects, and are suitable for biosafety level (BSL) 1 handling

how they work

CellLight® reagents are fusion constructs of signal peptides or cell structure proteins with premier emGFP, TagRFP, or CFP labels for accurate and specific targeting to subcellular compartments and structures. CellLight® reagents that target a variety of structures, organelles, and processes are available for convenient multiplexing, colocalization studies, and imaging of dynamic cellular processes where high spatial and temporal resolution is required. In addition, CellLight® reagents tolerate fixation with formaldehyde and therefore are compatible with fixed-cell analysis. We’ve just added three new CellLight® reagents to our portfolio.

 

CellLight® reagents 
Live-cell imaging of peroxisomes and endosomes using CellLight® reagents. HeLa cells were co-transduced with CellLight® Peroxisomes-RFP and CellLight® Early Endosomes-GFP and incubated overnight. (A) 40x magnification. (B) An enlarged region of the cell in panel A.



Quantify Autophagy—New Premo™ Autophagy TR-FRET LC3B Kits

what they are

Premo™ Autophagy TR-FRET LC3B kits employ time resolved–fluorescence resonance energy transfer (TR-FRET) technology to detect autophagy in cells expressing LC3B-GFP fusion protein, a Green Fluorescent Protein–fused marker for autophagic membranes. The kits are available in two different sizes and formats. The expression kit contains LC3B-GFP BacMam 2.0 reagent and matching terbium (Tb)–anti-LC3B antibody for expression and measurement of autophagy in your desired cell type. The antibody kit contains Tb–anti-LC3B antibody for use with cell lines that express LC3B-GFP.

what they offer

  • Quantitative autophagy measurement—easily and objectively compare conditions and experiments with truly quantitative data
  • Results consistent with historical data—pharmacological results of autophagy modulators wortmannin, PI-103, and tamoxifen were consistent with those obtained by traditional methods
  • More data in less time—helps save you time by delivering quantitative data in three addition-only steps

 how they work

Premo™ Autophagy Terbium/GFP TR-FRET LC3B Kits give you the ability to quantify autophagy. Autophagy activity is measured using cells expressing GFP-tagged LC3B combined with a terbium (Tb)-based TR-FRET immunoassay approach. This format provides you with an objective autophagy measurement. The assay uses a 96- or 384-well format, allowing speed and scalability not available with traditional autophagy methods such as western blot, fluorescence microscopy, and high-content analysis. 

 

The Premo™ Autophagy Tb⁄GFP TR-FRET LC3B Expression Kit 

The Premo™ Autophagy Tb/GFP TR-FRET LC3B Expression Kit.



Study CREB and Downstream Signaling Partners—New Antibodies for CREB and p38 MAPK

what they are

cAMP responsive element binding protein (CREB) is a transcription factor involved in up-regulation or down-regulation of many genes, depending on tissue type and phenotypic context. CREB is activated by secondary effectors such as divalent calcium ions and cyclic AMP, whose levels fluctuate with levels of growth or stress signals through MAPK, ERK, and p38 MAPK signaling cascades. ABfinity™ antibodies specific to CREB and downstream signaling cascade targets such as p38 MAPK are valuable tools for your signaling research.

what they offer

ABfinity™ recombinant monoclonal and oligoclonal antibodies offer consistent results, minimizing 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. Our CREB ABfinity™ Recombinant Rabbit Antibodies are validated for western blotting and immunocytochemistry applications. p38 MAPK [pTpY180/182] monoclonal and oligoclonal antibodies are validated for use in western blots and ELISAs. 

 

CREB Rabbit Recombinant Oligoclonal Antibody
Analysis of HeLa cells stained with CREB Recombinant Rabbit Oligoclonal Antibody. CREB in HeLa cells was evaluated by immunocytochemistry using CREB Recombinant Rabbit Oligoclonal Antibody followed by Alexa Fluor® 488 goat anti-rabbit secondary antibody (green). Nuclei were stained with DAPI (blue) and actin was stained with Alexa Fluor® 594 phalloidin (red). (A) Cells showing nuclear localization of CREB. (B) Cells stained with DAPI. (C) Merged image.
Antibody Target Host Antibody Type Quantity Cat. No.
CREBRabbitOligoclonal100 µg710149
p38 MAPK [pTpY180/182]RabbitMonoclonal100 µg701057
p38 MAPK [pTpY180/182]RabbitOligoclonal100 µg710088

NEW APPLICATIONS

Tissue Homeostasis and Detection of Apoptosis

CellEvent® Caspase-3/7 Green Detection Reagent is designed to detect caspase-3/7 activity—one of the hallmarks of apoptosis—with a single-step fluorescence assay, without requiring a wash step. CellEvent® reagent is fixable and cell permeant, making it useful for multiplex analysis as well as diverse imaging platforms such as in vitro imaging, high-content screening, high-throughput screening, fluorescence microplate readers, and flow cytometers.

Recently, Heather Brosnan and Dr. Philip Bickler from the Bickler lab, University of California, San Francisco, used CellEvent® Caspase-3/7 Green Detection Reagent for a new application—to detect apoptosis in tissue sections. The researchers prepared hippocampus tissue slices from 7-day-old Sprague-Dawley rats according to standard protocols [1,2] and maintained them in culture for 6 days. The sections were separated into two groups of five sections each. Both groups were loaded with 5 µM CellEvent® Caspase-3/7 Green Detection Reagent. After approximately 1 hour, one group was treated with 1 µM staurosporine to induce apoptosis, and the other group was left untreated as a control. As shown below, CellEvent® Caspase-3/7 Green Detection Reagent clearly labeled apoptotic cells throughout the entire tissue section.

References

  1. Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37:173–182.
  2. Sullivan BS, Leu D, Taylor DM et al. (2002) Isoflurane prevents delayed cell death in an organotypic slice culture model of cerebral ischemia. Anesthesiology 96:189–195

 

 CellEvent™ Caspase-3/7 Green Detection Reagent 
Apoptosis detection in rat hippocampus slice cultures using CellEvent® Caspase-3/7 Green Detection Reagent.
Organotypic cultures of rat hippocampus were prepared by standard methods [1,2]. CellEvent® Caspase-3/7 Green Detection Reagent was diluted to 5 µM in medium (75% Eagle’s Minimal Essential Medium without phenol red and 25% Earle’s Balanced Salt Solution). The assay was left to load for 1 hr prior to imaging. Staurosporine was used for a positive control at a concentration of 1 µM. One well was used per experimental group; each well contained five slices. Digital images of the slices were taken using a SPOT Jr. Digital Camera (Diagnostic Instruments, Sterling Heights, MI) and an inverted microscope. Fluorescence intensity was analyzed with Image J software, and the background fluorescent signal from non-slice regions of the images was subtracted from the total fluorescent signal in the slice region.
Figure generated by Heather Brosnan and Philip Bickler, MD, PhD, from the Bickler lab, University of California, San Francisco.

PROVEN PERFORMERS

Simultaneous Analysis of Cell Proliferation and Mitotic Arrest on the Tali™ Image-Based Cytometer

With its state-of-the-art optics and intuitive image-analysis software, the Tali™ Image-Based Cytometer is an ideal instrument for comparing proliferating cells to those in mitotic arrest. Click-iT® EdU assays offer direct measurement of nascent DNA to evaluate cell proliferation in a population. These assays use EdU (5-ethynyl-2´-deoxyuridine), a nucleoside analog of thymidine that is incorporated into DNA during active DNA synthesis. The nascent DNA is then labeled with small azide derivatives of fluorescent dyes via a copper-catalyzed covalent “click reaction” with the incorporated EdU residues. Because Click-iT® EdU assays are compatible with standard immunolabeling protocols, detection of cell proliferation can be multiplexed with chromosome condensation analysis using the HCS Mitotic Index Kit, which provides a primary antibody against histone H3 (pS10) as a sensitive index of mitosis, as well as a secondary antibody conjugated to the Alexa Fluor® 488 dye. Results of this suspension cell–based, two-color (GFP/RFP) assay can be viewed on the Tali™ cytometer.

Click-iT® EdU Assay for cell proliferation

  • Rapid—fast, highly sensitive method for detecting DNA synthesis
  • Compatible—can be used with standard immunolabeling protocols
  • Superior alternative—outperforms traditional BrdU assays
     

Tali™ Image-Based Cytometer for quick, quantitative cell analysis right at your bench

  • Accurate—obtain statistically significant three-parameter population analysis
  • Versatile—generate visual and analytical data for GFP/RPF expression, apoptosis, and cell viability
  • Convenient—set up in minutes with no cleaning and minimal maintenance

 

 

Click-iT® EdU Assay 
Analyzing cell proliferation and mitotic arrest on the Tali™ Image-Based Cytometer.
Jurkat cells were treated with 500 nM nocodazole for 20 hr at 37°C/5% CO2, or left untreated, and assayed using the Click-iT® EdU Alexa Fluor® 555 HCS Assay and the HCS Mitotic Index Kit, which includes anti–phosphohistone H3 primary antibody detected using
Alexa Fluor® 488 goat anti-rabbit secondary antibody (green). Segmentation and cell counting were conducted in transmitted light, and fluorescence data from Alexa Fluor® 555 azide dye (red) and Alexa Fluor® 488 dye (green) were obtained using GFP and RFP fluorescence channels. In the untreated control, there was strong Alexa Fluor® 555 dye staining, indicative of cell proliferation. Cells that were treated with 500 nM nocodazole showed a strong increase in phosphohistone H3 staining, indicative of mitotic arrest, in conjunction with a decrease in Alexa Fluor® 555 dye staining of proliferating cells.

DEPARTMENTS

On the Web

 

Your Image in 10 Minutes or Less

Watch our video to see how easy it is to capture a cell image less than 10 minutes after receiving the FLoid™ Cell Imaging Station. Designed in collaboration with fluorescence microscopists, the FLoid™ benchtop instrument features high-quality three-color image capture capability and an intuitive interface to help you collect data with just a few mouse clicks.

Imaging Corner

Detection of the Golgi Apparatus Using Tyramide Signal Amplification
 

Detection of the Golgi Apparatus Using Tyramide Signal Amplification (TSA™)

Formaldehyde-fixed and Triton® X-100–permeabilized BPAE cells were blocked with goat serum, nuclei were stained with DAPI (blue), and F-actin was stained with Alexa Fluor® 488 phalloidin (green). Anti–golgin-97 IgG1 was labeled using TSA™ Kit #5 with HRP–goat anti–mouse IgG and Alexa Fluor® 594 tyramide to indicate cell-to-cell adhesion (red).

From the Bench

Myosin Va and Myosin VI Coordinate Their Steps While Engaged in an In Vitro Tug of War During Cargo Transport
Ali MY, Kennedy GG, Safer D (2011) Proc Natl Acad Sci USA 23;108(34):E535-541.

Molecular motors not only are fascinating but also are involved in many biological processes, including intracellular transport of vesicles and biomolecules, cell division, cell movement, and signal transduction. In a recent study described in Proc Natl Acad Sci USA, M. Yusuf Ali and colleagues investigated intracellular transport mechanisms by two members of the myosin superfamily of molecular motors. The researchers studied transport by class V and VI myosin motors (myoV and myoVI), which walk along polarized actin filaments in opposite directions using a hand-over-hand motion. Since both myoV and myoVI colocalize to organelles, they may be involved in a biological tug of war if both are associated with the same cargo. To help elucidate how these motors can effectively transport their cargo under such circumstances, the authors developed “an in vitro model system in which a single myoV molecule was physically linked to an oppositely directed single myoVI through a common cargo in order to characterize the mechanical interactions during this virtual tug of war.” They labeled each motor with a different colored Qdot® nanocrystals and were able to determine “that myoV is the dominant motor and that, as myoVI resists, it steps backward in coordination with myoV’s forward steps.” They also found that “myoVI can prevent myoV from dominating by myoVI shifting its role from transporter to anchor in the presence of ADP, thus, potentially linking cargo transport to the cell’s metabolic state.”

Methods used in this research for labeling single-molecule motors with quantum dots are described in a title in the Methods in Molecular Biology series.

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