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Conferences & Tradeshows
Visit Molecular Probes and Invitrogen at conferences and trade shows. Listed below is the next meeting that we will be attending.
Keystone Symposia—Lymphocyte Activation and Gene Expression
Breckenridge, CO, USA
February 27–March 4, 2010
Keystone Symposia—Angiogenesis in Health and Disease
Keystone, CO, USA
February 28–March 5, 2010
ToxExpo
Salt Lake City, UT, USA
March 7–11, 2010
Keystone Symposia—Cell Death Pathways
Vancouver, British Columbia
March 12–17, 2010
Keystone Symposia—Lymphocyte Activation and Gene Expression
Breckenridge, CO, USA
February 27–March 4, 2010
Keystone Symposia—Angiogenesis in Health and Disease
Keystone, CO, USA
February 28–March 5, 2010
ToxExpo
Salt Lake City, UT, USA
March 7–11, 2010
Keystone Symposia—Cell Death Pathways
Vancouver, British Columbia
March 12–17, 2010
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In This Issue
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FEATURED PRODUCTS
NEW APPLICATIONS
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DEPARTMENTS
Check out the latest issue of BioProbes
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FEATURED NEW PRODUCTS
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One Anti-GFP Antibody, Multiple Applications—ABfinity™ Recombinant Rabbit Monoclonal Anti-GFP
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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
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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. |
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Characterize 10 Generations of Proliferating Cells—CellTrace™ Violet Cell Proliferation Kit
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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
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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. |
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Easier Detection of Secreted Alkaline Phosphatase—NovaBright™ SEAP Enzyme Reporter Gene Chemiluminescent Detection Assay Kit 2.0
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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
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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.
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NEW APPLICATIONS
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New Technique Exposes mtDNA Biogenesis
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.
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.
- Learn More about Click-iT® EdU
- Learn More about TSA® Technology and Kits
- Read the article
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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. |
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Product Quantity Cat. No. EdU (5-ethynyl-2'-deoxyuridine) 50 mg A10044 
Click-iT® Cell Reaction Buffer Kit 1 kit C10269 Oregon Green® 488 azide (Oregon Green® 6-carboxamido-(6-azidohexanyl), triethylammonium salt) *6-isomer* 0.5 mg O10180 Anti-fluorescein/Oregon Green®, rabbit IgG fraction, horseradish peroxidase conjugate 0.5 mg A21253 TSA® Kit #12 *with HRP–goat anti-rabbit IgG and Alexa Fluor® 488 tyramide* *50-150 slides* 1 kit T20922 Alexa Fluor® 594 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/mL* 0.5 mL A11032
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DEPARTMENTS
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Buzzworthy
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
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
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 | 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. |
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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]
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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. | |
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| MitoSOX™ Red Mitochondrial Superoxide Indicator, for live-cell imaging | 10 x 50 µg | M36008 | |
| References | |
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| 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. |
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 | 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.
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 | 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.
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 | 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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