ProbesOnline February 2010

In This Issue

FEATURED PRODUCTS

 
One Anti-GFP Antibody, Multiple Applications ABfinity™ Recombinant Rabbit Monoclonal Anti-GFP

 Characterize 10 Generations of Proliferating Cells CellTrace™ Violet Cell Proliferation Kit

 
Easier Detection of Secreted Placental Alkaline Phosphatase NovaBright™ SEAP Enzyme Reporter Gene Chemiluminescent Detection Kit 2.0

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


NEW APPLICATIONS


 
New Technique Exposes mtDNA Biogenesis

FEATURED NEW PRODUCTS

what it is
Why use different anti-GFP antibodies for different applications? Invitrogen’s new anti–Green Fluorescent Protein (GFP) antibody, ABfinity™ Recombinant Rabbit Monoclonal Antibody, is suitable for detecting GFP in multiple applications, including western blots, immunoprecipitation, ELISA, flow cytometry, immunohistochemistry, and immunocytochemistry. It’s a simple choice for all your GFP detection needs.




what it offers

  • A single anti-GFP antibody that does it all!
how it works
ABfinity™ antibodies are recombinant monoclonal antibodies developed by immunizing rabbits, screening for functionality, and cloning the immunogen-specific antibody genes into high-level expression vectors. The new anti-GFP antibody was raised against full-length GFP and can be used to detect native GFP, GFP variants, and most GFP fusions.
ER-GFP Organelle Lights
 
Anti-GFP ABfinity™ Recombinant Rabbit Monoclonal Antibody used for immunocytochemistry. (A) U2OS cells expressing Organelle Lights™ ER-GFP were incubated with the anti-GFP ABfinity™ Recombinant Rabbit Monoclonal Antibody. (B) Cells were formaldehyde-fixed, permeabilized, and blocked in 1% BSA, then incubated with primary antibody at 1 μg/mL, followed by Alexa Fluor® 647 goat anti–rabbit IgG conjugate. (C) The merged yellow signal indicates colocalization of GFP fluorescence and the detection antibody. Nuclei were stained with Hoechst 33342.

View larger image for more details
Product
Quantity Cat. No.
GFP, ABfinity™ Recombinant Rabbit Monoclonal Antibody—unconjugated 100 μg G10362Order Now
    
what it is
Amine-reactive cell tracing dyes, such as the new CellTrace™ Violet, are known to provide excellent long-term retention in cells. CellTrace™ Violet is a cell-permeant dye that enters live cells, where it is converted to a fluorescent derivative by nonspecific esterases. The resulting succinimidyl ester covalently binds to amine groups in proteins, resulting in long-term dye retention.



what it offers

  • Bright, homogenous staining—visualization of 10 or more generations of proliferating cells
  • Long-term signal stability—consistent signal, even after several days in culture
  • Ease of multiplexing—can be used with most blue-, green-, or red-excitable fluorophores
  • Nontoxic—labeled cells maintain high viability, even after 7 days in culture
how it works
When a cell labeled with CellTrace™ Violet divides, each daughter cell receives approximately half of the fluorescent label, the next generation receives a quarter, and so on. Analysis of the fluorescence intensities of cells labeled and grown in vivo or in vitro enables determination of the number of generations through which a cell has progressed since the label was applied. The kit allows you to identify dividing cells in biological systems.
CellTrace Violet Cell Proliferation
 
Visualization of 10 generations of T lymphocytes using the CellTrace™ Violet Proliferation Kit. The peaks represent successive generations of human CD8+ T lymphocytes stimulated with 200 ng mouse anti–human CD3 and 100 ng interleukin-2 per milliliter and incubated in OpTmizer™ T-cell Expansion Medium at 37°C for 7 days. The parent peak represents cells that were grown in culture for 7 days with no stimulus.

View larger image for more details
Product
Unit Size Cat. No.
CellTrace™ Violet Cell Proliferation Kit180 tests C34557Order Now
CellTrace™ CSFE Proliferation Kit—for flow cytometry1 kit C34554Order Now
Purified Mouse Anti-Human CD3500 µL MHCD0300Order Now
Recombinant Human Interleukin-210 µgPHC0026Order Now
OpTmizer™ T-Cell Expansion SFM500 mL 080022SDOrder Now
    
what it is
The NovaBright™ Secreted Placental Alkaline Phosphatase (SEAP) 2.0 kit makes detection of SEAP reporter gene activity easier than ever. The procedure has been simplified down to two ready-to-use assays—reagent preparation and sample dilution steps have been eliminated!



what it offers

  • Sensitivity—beats detection by fluorescence or colorimetric methods
  • Assay simplicity—two ready-to-use reagents, no sample dilutions
  • Improved signal-to-noise ratio and low-end sensitivity

how it works
The kit combines our high-performance alkaline phosphatase substrate, CSPD®, and our next-generation Emerald™ enhancer to provide better assay performance over 5 orders of magnitude of enzyme concentration.  Our proprietary buffer formulation minimizes the background due to endogenous phosphatases, enabling the NovaBright™ SEAP 2.0 kit to provide the most sensitive detection with a higher signal-to-noise ratio of SEAP reporter gene activity than other assays of its kind.


NovaBright SEAP Assay
 
NovaBright™ SEAP 2.0 assay kinetics compared to other SEAP reporter gene kits. pCMV-transfected NIH/3T3 cells were assayed with NovaBright™ SEAP 2.0 (blue), NovaBright™ SEAP (red), supplier A (green), or supplier B (orange) assay kits. The NovaBright™ SEAP and SEAP 2.0 kits enable detection of secreted placental alkaline phosphatase with lower limits of detection and better signal-to-noise ratios than the other commercially available kits.

View larger image for more details
Product
Quantity Cat. No.
NovaBright™ SEAP Enzyme Reporter Gene
Chemiluminescent Detection Kit 2.0
192 assays
N10577Order Now
NovaBright™ SEAP Enzyme Reporter Gene
Chemiluminescent Detection Kit 2.0
960 assays
N10578Order Now
 

NEW APPLICATIONS

Detecting Mitochondrial Biogenesis
Mitochondrial biogenesis enables neurons to meet changing energy loads and to redistribute mitochondria throughout the neuron. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. Traditional methods for monitoring nascent nuclear DNA synthesis follow the incorporation of a nucleoside analog of thymidine into DNA—involving either radioactive 3H-thymidine or BrdU. Direct visualization of mtDNA synthesis can be challenging due to the weak signal obtained from the small mitochondrial genome (~17 kb); inhibiting nuclear DNA replication does not increase sensitivity.


New Technique
The easy-to-use Click-iT® EdU assay, when combined with Tyramide Signal Amplification (TSA®) technology, enables sensitive and reliable measurement of nascent mtDNA synthesis. The Click-iT® EdU assay is much easier to perform, eliminating the use of radioactivity and avoiding the harsh DNA denaturation step required with the BrdU method, while TSA® technology has been reported to increase detection sensitivity up to 100-fold compared to conventional avidin-biotinylated enzyme complex (ABC) procedures.

TSA® is an enzyme-mediated detection method that uses the catalytic activity of horseradish peroxidase (HRP) to achieve high-density labeling of a target antigen. For the mtDNA detection assay, the target antigen is the fluorescent azide used in the Click-iT® EdU assay to detect DNA containing incorporated EdU. An HRP-conjugated anti-dye antibody is then combined with a tyramide analog that has the same fluorescence emission as the original fluorescent azide.





TSA & Click-iT EdU

mtDNA replication with TSA® and Click-iT® EdU in dissociated dorsal root ganglion neurons.
Neurons plated on glass coverslips were incubated with 10 μM EdU. Following fixation and permeabilization, endogenous peroxidase activity was blocked and EdU was detected with Oregon Green® 488 azide. The Oregon Green® 488 signal was amplified using HRP-conjugated rabbit antibody against Oregon Green® 488 and Alexa Fluor® 488 tyramide from TSA® Kit #12. The pan-neuronal marker αTuj1 was detected with a mouse primary antibody and visualized with an Alexa Fluor® 594 goat anti-mouse secondary antibody. Image contributed by Stephen I. Lentz and Eva L. Feldman, Departments of Internal Medicine and Neurology, University of Michigan.
Product
Quantity Cat. No.
EdU (5-ethynyl-2'-deoxyuridine)50 mg A10044Order Now
Click-iT® Cell Reaction Buffer Kit1 kitC10269Order Now
Oregon Green® 488 azide (Oregon Green® 6-carboxamido-(6-azidohexanyl), triethylammonium salt) *6-isomer*0.5 mg O10180Order Now
Anti-fluorescein/Oregon Green®, rabbit IgG fraction, horseradish peroxidase conjugate0.5 mg A21253Order Now
TSA® Kit #12 *with HRP–goat anti-rabbit IgG and Alexa Fluor® 488 tyramide* *50-150 slides*1 kitT20922Order Now
Alexa Fluor® 594 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/mL*0.5 mL A11032Order Now
    

DEPARTMENTS

Labeling and tracking of mesenchymal stromal cells with EdU.
Lin G et al. (2009) Cytotherapy 11:864–873.

Can EdU-based labeling be used to track stem cell activity in vivo?
The reactive nucleoside analog EdU is gaining widespread acceptance as an alternative to BrdU for the visualization of proliferating cells, owing largely to the milder conditions required for its use. While EdU has demonstrated effective labeling in a wide variety of cell types, its utility for tracking stem cells has not been shown.

Methods
In their recent report, Lin and colleagues use EdU-based Click-iT® labeling technology with detection by Alexa Fluor® 594 azide to label and track adipose tissue–derived stem cells (ADSCs) in culture and in living animals.

Results

EdU labeling under culture conditions was observed only in the nuclei of replicating ADSCs; EdU-positive cells remained visible through 21 days of observation. Tissues removed from rats that had received EdU intraperitoneally were successfully stained with Alexa Fluor® 594 azide, with particularly intense fluorescence observed in kidney, lung, and intestine. Further, the group transplanted in vitro–labeled ADSCs subcutaneously into rats and observed persistent fluorescence through 6 weeks of observation. Transplantation of in vitro–labeled ADSCs into rats suffering from a hyperlipidemia-associated bladder disorder revealed the presence of EdU-positive cells in bladder connective tissue at 4 and 10 weeks post-transplantation. In contrast, previously reported results suggest BrdU might only be effectively tracked for as little as 2 weeks under similar circumstances.

Conclusion
These results demonstrate the utility of Click-iT® EdU–based labeling as a method for tracking transplanted stem cells, which could provide an effective observational methodology in preclinical settings. 

View the bibliography reference

Learn More about Click-iT® EdU Assays



  Visualizing Gustatory Neurons in a Mouse Tongue. This wild type mouse embryo was fixed in 4% phosphate-buffered paraformaldehyde, and taste ganglia were labeled with the lipophilic tracer DiI. Images were taken with an Olympus confocal microscope. Image provided by Dr. Robin F. Krimm, Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine.




Proven Performers—MitoSOX™ Red Mitochondrial Superoxide Indicator: A Versatile Probe Across Applications

The superoxide anion reactive oxygen species (ROS), generated as a by-product of mitochondrial oxidative phosphorylation, is a pervasive source of toxicity and mitochondrial dysfunction. The enzyme superoxide dismutase (SOD) performs the key function of counteracting excessive levels of superoxide by converting it to less-reactive hydrogen peroxide.

MitoSOX™ Red Superoxide Indicator, a derivative of dihydroethidium (also known as hydroethidine), undergoes hydroxylation when oxidized by superoxide to produce a 2-hydroxyethidium derivative that exhibits a fluorescence excitation peak at ~400 nm. This peak is absent in the excitation spectrum of the ethidium oxidation product generated by ROS other than superoxide. Thus, fluorescence excitation at 400 nm with emission detection at ~590 nm provides optimum discrimination of superoxide from other ROS. Many of the applications of MitoSOX™ Red indicator for specific detection of mitochondrial superoxide in live cells and tissues revolve around SOD and Complex I, disorders in their expression, structure, and function, and their ultimate consequences in various pathologies. The versatility of MitoSOX™ Red indicator has been proven in diverse applications, including:

  • Neuroscience—one of several probes used to provide a correlative assessment of multiple physiological parameters [1]
  • Cardiovascular biology—used in combination with Amplex® Red reagent, for measurement of mitochondrial superoxide and hydrogen peroxide production in rat vascular endothelial cells [2]
  • Flow cytometry—Mukhopadhyay et al. [3] have published detailed protocols for simultaneous measurement of mitochondrial superoxide generation and apoptotic markers (allophycocyanin–annexin V and SYTOX® Green stain) in human coronary artery endothelial cells by flow cytometry
  • Oxidative damage—quantitative imaging of MitoSOX™ Red indicator has shown that treatment of vascular endothelial cells with extensively oxidized low-density lipoprotein (LDL) resulted in near doubling of mitochondrial superoxide generation compared to treatment with normal LDL [4]


MitoSOX Red Superoxide Indicator

Detecting superoxide in live cells using MitoSOX™ Red Superoxide Indicator.
Live 3T3 fibroblasts were treated with iron porphyrin FeTCPP, a superoxide dismutase (SOD) mimetic (B), or left untreated (A). Cells were then labeled with MitoSOX™ Red indicator in combination with blue-fluorescent Hoechst 33342 nuclear stain. Knockdown of the mitochondrial fluorescence signal by SOD mimetics such as FeTCPP and the manganese porphyrin MnTBAP are valuable negative controls in MitoSOX™ Red superoxide detection experiments. Useful positive controls (not shown) include treatment with antimycin A, doxorubicin, or high glucose.


Product
Quantity Cat. No.
MitoSOX™ Red Mitochondrial Superoxide Indicator, for live-cell imaging
10 x 50 µg
M36008Order Now
    

References
1. Quintanilla RA, Matthews-Roberson TA, Dolan PJ et al. (2009) J Biol Chem 284:18754–18766.
2. Ungvari Z, Labinskyy N, Gupte S et al. (2008) Am J Physiol Heart Circ Physiol 294:H2121–H2128.
3. Mukhopadhyay P, Rajesh M, Haskó G et al. (2007) Nat Protoc 2:2295−2301.
4. Roy Chowdhury SK, Sangle GV, Xie X et al. (2010) Am J Physiol Endocrinol Metab 298:E89–E98.
  


 Improved Selection Guide for Flow Cytometry Antibodies

We've made it much easier to find the exact antibody you need for your flow cytometry experiments. Choose to browse by CD molecule, cell type, or antibody function. You can then further refine your result by filtering by conjugate type. All of the antibodies in this selection guide have been validated for flow cytometry, so you can be confident in your results.





 Improved Qubit™ Quantitation Platform Webpage

The Qubit™ Quantitation Platform combines the small and economical Qubit® Fluorometer with highly sensitive fluorescence-based Quant-iT™ assays to provide seamless protocols for DNA, RNA, and protein quantitation. The system is simple, fast, and easy to use, yet consistently produces accurate results so that you can be confident moving forward with subsequent applications. With our improved web resource, it’s now easier to view all Qubit™ technical data and the latest technical notes. You can also conveniently download the latest publications and frequently asked questions.





 iPhone® Applications & Widgets

Invitrogen now offers an assortment of handy widgets, and will soon be adding iPhone® apps, for your everyday needs. With so many research tools available, including the interactive and comprehensive Alexa Fluor® Selection Guide widget and the convenient Everyday Science Calculators app, you’ll find many quick ways to get the information you need. Visit often, as we'll be adding new widgets and apps as they become available.




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