ProbesOnline January 2010

In This Issue

FEATURED PRODUCTS

 
Absolute Detection of Apoptotic Transformation Violet Ratiometric Membrane Asymmetry/Dead Cell Apoptosis Kit

 
Dual Pulse Labeling of Cell Proliferation New BrdU Antibodies and Click-iT® EdU

New Products for Cell &Tissue Analysis
 
See all of this month's New Products for Cell & Tissue Analysis


NEW APPLICATIONS


 
It’s Time to Replace 3H-Thymidine With Click-iT® EdU

FEATURED PRODUCTS

what it is
The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit provides an easy, efficient method for the detection of apoptosis using a violet laser flow cytometer. The assay is compatible with adherent and suspension cell types and correlates with other indicators of apoptosis, including caspase activation and changes in mitochondrial membrane potential.



what it offers

  • Robust assay—ratiometric probe is independent of probe concentration, cell size, and instrument variation
  • Easy to use—5-minute incubation, with no wash required
  • Flexible experimental design—easily combined with other testing reagents
how it works
This assay employs a novel dye (F2N12S) that incorporates selectively into the outer leaflet of the plasma membrane. The dye exhibits dual fluorescence emission bands corresponding to 530 nm and 585 nm, producing a ratiometric response to variations in plasma membrane surface charge during early apoptosis that is detected by flow cytometry. The kit also includes SYTOX® AADvanced™ Dead Cell Stain for discrimination of dead cells from live cells. Unlike annexin V conjugates, this assay requires no special buffers or wash steps and is less affected by cell membrane damage commonly associated with the removal of adherent cells, thereby improving data quality.
Product
Quantity Cat. No.
Violet Ratiometric Membrane AsymmetryProbe/Dead Cell Apoptosis Kit *for flow cytometry*
1 kit A35137Order Now
    
what they are
BrdU mouse monoclonal antibodies (clone MoBU-1) are now available for use in flow cytometry and imaging applications, including BrdU cell proliferation assays, BrdUTP TUNEL assays, and dual pulse cell proliferation assays with Click-iT® EdU.



what they offer

  • High specificity for BrdU 
  • No cross-reactivity with EdU
  • Simplified, straightforward, and reliable method for dual pulse labeling

how they work
The traditional method of measuring cell proliferation employs the incorporation of the thymidine analog BrdU into newly synthesized DNA, and its subsequent detection with an anti-BrdU antibody. Although this method is rapidly being replaced with the click chemistry–based Click-iT® EdU assay, an advantage of the antibody clone MoBU-1 is that it does not cross-react with the thymidine analog EdU, thus making it valuable for use in dual pulse cell proliferation detection in combination with EdU labeling.
Product
Unit Size Cat. No.
BrdU mouse monoclonal antibody (Clone MoBU-1) unconjugated350 µLB35128Order Now
BrdU mouse monoclonal antibody (Clone MoBU-1) Alexa Fluor ® 488 *0.2 mg⁄mL*350 µL B35130Order Now
BrdU mouse monoclonal antibody (Clone MoBU-1) Alexa Fluor ® 594 *0.2 mg⁄mL*350 µL B35132Order Now
BrdU mouse monoclonal antibody (Clone MoBU-1) Alexa Fluor ® 647 *0.2 mg⁄mL*350 µL B35133Order Now
BrdU mouse monoclonal antibody (Clone MoBU-1) Pacific Blue™ *for flow cytometry* 100 tests B35129Order Now
BrdU mouse monoclonal antibody (Clone MoBU-1) Alexa Fluor® 488 *for flow cytometry* 100 tests B35139Order Now
BrdU mouse monoclonal antibody (Clone MoBU-1) Alexa Fluor® 647 *for flow cytometry* 100 tests B35140Order Now
    

NEW APPLICATIONS

For several decades, 3H-thymidine has been the gold standard for measuring T lymphocyte proliferation. This method has been generally replaced with antibody-based detection of the nucleoside bromodeoxyuridine, with the understanding that eliminating the radioactivity of 3H-thymidine would come at a cost—the harsh DNA denaturation step required with the BrdU method, a step that can be lengthy, difficult to perform consistently, and most importantly, can destroy sample integrity, including cell morphology and other antigen-recognition sites. The Click-iT® EdU cell proliferation assay not only eliminates the use of radioactivity but does not require any DNA denaturation for detection of the incorporated nucleoside.  

Until now, the Click-iT® EdU method has only been compared to the BrdU technique. Direct evidence is now available that clearly demonstrates that Click-iT® EdU “strongly correlates with 3H-thymidine incorporation into proliferating T cells, and distinguishes between low, moderate, and robust T cell responses.” (Yu et al. (2009) J Immunol Methods 350:29–35).


 Cell Proliferation 

Cell proliferation in the mid-intestine of a zebrafish larva.
A 5-day-old zebrafish larva was exposed to a 16 hr pulse of 400 μM EdU, fixed, and processed for detection using the Click-iT® EdU Alexa Fluor® 488 Imaging Kit. Slides were subsequently stained with Alexa Fluor® 568 soybean agglutinin (SBA) and TO-PRO®-3 dye. Proliferating nuclei appear white due to labeling with both Alexa Fluor® 488 azide (green) and TO-PRO®-3 dye (blue). Goblet cells in the intestinal bulb, labeled with Alexa Fluor® 568 SBA, appear red. Image submitted by Sarah Cheesman, Institute of Molecular Biology, University of Oregon, USA.
Product
Quantity Cat. No.
Click-iT® EdU Alexa Fluor® 488 Imaging Kit1 kit, 50 coverslipsC10337Order Now
Click-iT® EdU Alexa Fluor® 555 Imaging Kit1 kit, 50 coverslipsC10338Order Now
Click-iT® EdU Alexa Fluor® 594 Imaging Kit1 kit, 50 coverslipsC10339Order Now
Click-iT® EdU Alexa Fluor® 647 Imaging Kit1 kit, 50 coverslipsC10340Order Now
Click-iT® EdU Pacific Blue™ Flow Cytometry Assay Kit1 kit, 50 assaysA10034Order Now
Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit1 kit, 50 assaysC35002Order Now
Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit1 kit, 50 assaysA10202Order Now
    

DEPARTMENTS

p53-cofactor JMY is a multifunctional actin nucleation factor.
Zuchero JB et al. (2009) Nat Cell Biol 11:451–459.

What cellular factors regulate the assembly and function of actin structures?
Actin filaments are the basic building blocks for a wide array of cytoskeletal structures. Several classes of proteins have been identified that direct the assembly of these building blocks into macromolecular domains, including Arp2/3, which serves as a template for branched filament formation, and Spire, which nucleates unbranched filament assembly. In their current report, Zuchero and colleagues investigate the actin assembly role of JMY, a protein previously known as a transcriptional activator.

Methods
The effect of both JMY overexpression and mutation on actin organization was visualized in vivo in several cell types using Alexa Fluor® 568 phalloidin—a high-affinity probe for F-actin that is made from a mushroom toxin conjugated to the Alexa Fluor® 568 dye.

Results
JMY was capable of accelerating actin assembly in a dose-dependent fashion. Sequence comparison revealed homology between JMY and Spire; kinetic assays revealed the two proteins have nearly identical actin nucleation activities. JMY is largely localized in the nucleus of relatively nonmotile cells, but is found more prominently at the leading edge of growth regions in highly motile cells such as neutrophils, suggesting a role in cell motility. Subsequent experiments showed significantly reduced rates of healing in cells where JMY expression was suppressed by knockdown.

Conclusion
JMY is demonstrated to represent a new class of actin assembly factor with a critical role in cell motility.

View the bibliography reference

Product
Quantity Cat. No.
Alexa Fluor® 568 phalloidin300 units A12380Order Now
Alexa Fluor® 488 phalloidin300 units A12379Order Now
Lipofectamine® LTX Reagent15 mL 15338500Order Now
Alexa Fluor® 488 goat anti–rabbit IgG (H+L) *highly cross-adsorbed*0.5 mL A11034Order Now
    



 Madin-Darby Ovine Kidney Epithelial Cell 

Madin-Darby ovine kidney (MDOK) epithelial cell stained with Alexa Fluor® 350 conjugated to concanavalin A (blue), which selectively binds to α-mannopyranosyl and α-glucopyranosyl residues primarily in the ER. Actin filaments were stained with Alexa Fluor® 488 phalloidin (green). The nucleus was stained with SYTOX® Orange nuclear stain (orange). Cells were fixed and permeabilized prior to staining. Image courtesy of Dr. Michael W. Davidson, National High Magnetic Field Laboratory (NHMFL), The Florida State University.


Proven Performers — Durable Dyes Make Enduring Images

Alexa Fluor® fluorescent dyes, now in their 13th year of use, are well regarded for their brightness and extended durability under strong light exposure. The use of antifade reagents like our ProLong® Gold mountant can extend Alexa Fluor® dye photostability on fixed samples. However, this is not an option when viewing live samples or when imaging large whole-mount specimens. Recently, Keller et al. [1–4] demonstrated the exceptional photostability of Alexa Fluor® 546 secondary antibodies bound to β-tubulin primary antibodies in the absence of any antifade reagent in a whole-mount specimen. 

The group reconstructed the entire nervous system of the medaka fish in three dimensions by collecting over 50,000 separate scans using scanned light sheet illumination. The images show no evidence of Alexa Fluor® 546 photobleaching.

“Indeed, I did not observe any photobleaching at all…I can't see a difference in brightness between the first angle and the last angle at all, which is quite interesting, considering that 50,000 images of the embryo had already been collected by that time.”  — PJ Keller

The Alexa Fluor® 546 dye is ideal for achieving superior photostability in live-cell experiments in addition to whole mounts. Alexa Fluor® dyes also have the advantage of minimizing phototoxic by-products.

Secondary Antibodies

View larger image
 
Medaka fish nervous system imaged with Alexa Fluor® 546 secondary antibody. Fixed and permeabilized whole mount of a medaka fish was reacted with primary antibodies for β-tubulin followed by Alexa Fluor® 546 goat anti–mouse IgG. The stained whole embryo was scanned 2,100 times at each of 24, 15-degree rotations to create a 3D image. Over 50,000 scans were collected to create this enduring image with no detectable change in staining intensity. Image courtesy of Drs. P. Keller, A. Schmidt, J. Wittbrodt, and E. Stelzer of the European Molecular Biology Lab (EMBL) in Heidelberg, Germany.

 

References
1. Huisken J, Swoger J, Del Bene F et al. (2004) Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305:1007–1009.
2. Verveer PJ, Swoger J, Pampaloni F et al. (2007) High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy. Nat Methods 4:311–313.
3. Keller PJ, Schmidt AD, Wittbrodt J et al. (2008) Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322:1065–1069.
4. Keller PJ, Stelzer EH (2009) Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy. Curr Opin Neurobiol 18:1–9.
 

 Discover the Biological Applications of Click Chemistry
Click chemistry enables the specific labeling of DNA, RNA, viruses, proteins, and modified proteins in complex biological environments. Visit the new click biology web pages to discover how click chemistry can empower your experiments in cell proliferation, apoptosis, transcription, and translation to yield better results in much less time.



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