ProbesOnline August 2009

In This Issue

FEATURED NEW PRODUCTS

 
Ultrasensitive Reporter Gene AssaysNovaBright™ Reporter Gene Assays

 
Click-iT® Biology Just Got Brighter New Alexa Fluor® 555 Click-iT® EdU Cell Proliferation Assays and Detection Reagents

 
Live-Cell Imaging of Cell Cycle and DivisionPremo™ FUCCI Cell Cycle Sensor


 
Accelerate MAPK Signal Transduction ResearchNew Validated Primary Antibodies


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


NEW APPLICATIONS

 
Imaging Mitochondrial Calcium Sequestration — Organelle Lights™ Fluorescent Proteins Meet Classic Molecular Probes® Organelle Stains

FEATURED NEW PRODUCTS

what it is
Reporter gene assays are invaluable for the study of gene expression. NovaBright™ Reporter Gene Assays, available for the most common reporter genes, offer chemiluminescent measurement of reporter gene activity that is more sensitive than colorimetric or fluorescent detection. NovaBright™ kits are optimized to yield consistent results with high sensitivity over a wide dynamic range spanning femtogram to nanogram amounts of reporter.



what it offers

  • High sensitivity
  • Wide dynamic range
  • Three easy-to-use formats

how it works
NovaBright™ assays are offered in three easy-to-use formats:

  1. NovaBright™ Secreted Placental Alkaline Phosphatase (SEAP) Kit
  2. NovaBright™ β-galactosidase and Firefly Luciferase Dual Enzyme Reporter Gene Chemiluminescent Detection Kit
  3. NovaBright™ β-galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kit 

 

Product Quantity Cat. No.  
NovaBright™ Secreted Placental Alkaline Phosphatase (SEAP) Enzyme Reporter Gene Chemiluminescent Detection Kit400 assaysN10559Order Now
NovaBright™ Secreted Placental Alkaline Phosphatase (SEAP) Enzyme Reporter Gene Chemiluminescent Detection Kit1,200 assaysN10560Order Now
NovaBright™ β-galactosidase and Firefly Luciferase Dual Enzyme Reporter Gene Chemiluminescent Detection Kit200 assaysN10561Order Now
NovaBright™ β-galactosidase and Firefly Luciferase Dual Enzyme Reporter Gene Chemiluminescent Detection Kit600 assaysN10562Order Now
NovaBright™ β-galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kit for Mammalian Cells200 assaysN10563Order Now
NovaBright™ β-galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kit for Mammalian Cells1,000 assaysN10564Order Now
NovaBright™ β-galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kit for Yeast Cells200 assaysN10565Order Now
NovaBright™ β-galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kit for Yeast Cells1,000 assaysN10566Order Now

what it is
The Alexa Fluor® 555 dye, a superior alternative to tetramethylrhodamine (TRITC) and Cy®3 red-orange fluorophores, is now available as a detection reagent in click chemistry–based detection of cell proliferation and azide/alkyne-labeled biomolecules.



what it offers

  • Simply the best fluorescence—brighter and more photostable than TRITC and Cy®3 dyes
  • Compatible—enables multiplexed analyses with blue, green, and far-red–fluorescent dyes
  • Click-iT® biology—superior alternative to radioactivity- and antibody-based detection

how it works
Click-iT® biology utilizes biologically unique moieties for efficient labeling and detection of molecules of interest using a simple, two-step procedure. In the first step, an azide- or alkyne-containing biomolecule is fed to cells. Unlike other labels, the azide and alkyne tags are small enough that tagged biomolecules are acceptable substrates for incorporation into macromolecules, including proteins, DNA, or RNA. The subsequent detection step utilizes the chemoselective “click” ligation reaction between an azide and alkyne where the modified biomolecule is detected with a corresponding azide- or alkyne-containing dye or hapten. Unlike antibodies (MW ~150 kDa), the Alexa Fluor® 555 Click-iT® detection molecule (MW ~1,000 Da) easily penetrates complex samples.

Alexa Fluor® 555 Click-iT® EdU Cell Proliferation Assay

Alexa Fluor® 555 Click-iT® EdU Cell Proliferation Assay. HeLa cells were treated for 30 min with EdU. Cells were fixed and permeabilized, and EdU that had been incorporated into nascent DNA was detected with the red-orange Alexa Fluor® 555 azide from the Click-iT® EdU 555 Imaging Kit. Tubulin was labeled with a mouse anti-tubulin antibody and visualized with the far-red–fluorescent Alexa Fluor® 647 goat anti-mouse antibody.
Product Quantity Cat. No.  
Alexa Fluor® 555 azide, triethylammonium salt0.5 mgA20012Order Now
Alexa Fluor® 555 alkyne, triethylammonium salt0.5 mgA20013Order Now
Click-iT® EdU Alexa Fluor® 555 Imaging Kit *for 50 coverslips*1 kitC10338Order Now
Click-iT® EdU Alexa Fluor® 555 HCS Assay *2-plate size*1 kitC10352Order Now
Click-iT® EdU Alexa Fluor® 555 HCS Assay *10-plate size*1 kitC10353Order Now
what it is
The Premo™ FUCCI cell cycle sensor enables live-cell imaging of cell cycle and division. As cells progress through the cell cycle, nuclear fluorescence changes from red to green.



what it offers
  • Ready-to-use—no need to purify plasmids or worry about vector integrity
  • Cell-friendly—no harmful treatments are required; primary and stem cells are labeled without apparent cytopathic effects 
  • Titratable—defined reagent concentrations enable precise titration and optimization of expression levels
how it works
In 2008, Miyawaki and colleagues developed the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), a fluorescent protein–based sensor that employs red (RFP) and green (GFP) fluorescent proteins fused to different regulators of the cell cycle: Cdt1 and geminin. The temporal regulation of these proteins by ubiquitin results in the biphasic cycling through the cell cycle. Premo™ FUCCI Cell Cycle Sensor takes this technology one step further by using the BacMam gene delivery system—the prepackaged genetically encoded reagents are ready for immediate use. Simply add the Premo™ FUCCI reagents to your cells, treat with the BacMam enhancer, wash, incubate overnight for protein expression, and visualize cell cycle progression in populations of cells using fluorescence microscopy.

Imaging cell cycle progression in live cells


Imaging cell cycle progression in live cells with Premo™ FUCCI Cell Cycle Sensor. (A) Schematic of cell cycle progression with nuclear fluorescence changes. (B) U2OS cells were transduced with Premo™ FUCCI Cell Cycle Sensor, then stained with Alexa Fluor® 647 wheat germ agglutinin.

Product Quantity Cat. No.  
Premo™ FUCCI Cell Cycle Sensor1 kitP36232Order Now

what it is
The mitogen-activated protein kinase (MAPK) pathway mediates signal transduction from cell surface receptors to downstream transcription factors that regulate cellular responses such as cell proliferation, growth, motility, survival, and apoptosis. Invitrogen’s broad portfolio of products for MAPK research includes both phosphospecific and total antibodies that are validated for multiple applications including western blots, immunoprecipitation, immunocytochemistry, and immunohistochemistry.



what it offers

  • Confidence— all phosphorylation site–specificities have been verified with peptide competition
  • Performance—robust antibodies validated for multiple applications
  • Selection—large menu of unique specificities, with wide range of targets and modification sites

how it works
The role of the MAPK pathway in cancer, immune disorders, and neurodegenerative diseases has been well recognized. Invitrogen now offers phosphospecific antibodies against MEK3, MEK4, and MEK6. MEK3 and MEK6 are members of a tyrosine/threonine protein kinase family that activate p38, which is part of the inflammation/stress signaling pathway. Phosphorylation of MEK3 and MEK6 by MEKK1 activates the proteins and enables them to phosphorylate p38. MEK4 is a member of a tyrosine⁄threonine protein kinase family that activates the c-Jun NH2-terminal kinases (JNK), which is also part of the inflammation⁄stress signaling pathway.

Phosphospecificity of the MEK3/6 antibody
 

Phosphospecificity of the MEK3/6 antibody. See larger image for more details
Product Species Reactivity* Applications† Quantity Cat. No.  
MEK1Hu, Ms, Rt, XWB, IP100 µg133500Order Now
MEK1 [pS298]Hu, Ms, RtWB10 blots44460GOrder Now
MEK2 [pT394]HuWB10 blots44466GOrder Now
MEK3/6 [pS189/pT193]/[207/211]HuWB10 blots44470GOrder Now
MEK4 [pS257/pT261]Hu (Ms)WB10 blots44474GOrder Now
MEK7 [pS271/pT275]Hu (Ms)WB10 blots44478Order Now
p38 MAPKHu, MsWB100 µgAHO1202Order Now
p38 MAPK [pT180/pY182]Hu, Rt (Ms, Cn, Mk)WB, IHC, ICC10 blots44684GOrder Now

* Cn = canine, Hu = human, Mk = monkey, Ms = mouse, Rt = rat, X = Xenopus. ( ) = reactivity predicted but not tested.
† ICC = immunocytochemistry, IHC = immunohistochemistry, IP = immunoprecipitation, WB = western blot.

NEW APPLICATIONS

The preferential accumulation of the calcium sensor rhod-2, AM in mitochondria has long been utilized to measure calcium flux, an important parameter given the vital role of mitochondria in maintaining intracellular calcium homeostasis. A limitation of using rhod-2 alone is that mitochondria are only visible after calcium uptake; however, determining mitochondrial location prior to the sequestration of calcium can provide important information about the positioning of individual mitochondria with respect to spatially localized intracellular calcium release. For more accurate measurement of mitochondrial calcium flux, the rhod-2 calcium indicator can be combined with probes for observing mitochondrial morphology, such as Organelle Lights™ Mitochondria GFP, for enhanced calcium imaging in individual mitochondria.

Organelle Lights™ reagents are ready-to-use fluorescent protein constructs fused with signal peptides for accurate and specific targeting to sub-cellular compartments and structures. Using the passive mitochondrial marker Organelle Lights™ Mito-GFP, heterogeneities in mitochondrial uptake and important mitochondrial dynamics such as fission, fusion, and motility can be observed before, during, and after the uptake of calcium into mitochondria. Pairing the green-fluorescent Organelle Lights™ Mito-GFP with the red-orange–fluorescent rhod-2, AM calcium sensor allows the spatial and temporal aspects of mitochondrial calcium sequestration to be analyzed with respect to individual mitochondrial dynamics within a given cell and across populations of cells.

 

Imaging mitochondrial calcium levels
Imaging mitochondrial calcium levels and dynamics with rhod-2, AM and Organelle Lights™ Mito-GFP. (A) HeLa cells were labeled with Organelle Lights™ Mito-GFP and treated with 5 µM rhod-2, AM for 15 min at 37°C. (BI) The region outlined in A is enlarged to show individual mitochondria within a single cell. (BII–III) Calcium release from internal stores following application of 10 µM histamine. Mitochondria in close proximity to the calcium release from internal stores are shown by the increase in the orange-red fluorescence of rhod-2, AM. The arrow in BII denotes a mitochondrion that has impaired mitochondrial calcium uptake, a detail that would not have been revealed using rhod-2, AM alone. The asterisk marks a previously motile mitochondrion that appears to have stopped moving following calcium elevation. Organelle Lights™ Mito-GFP and rhod-2, AM were imaged using standard FITC and TRITC filters, respectively, on a DeltaVision® Core microscope with a 40x objective lens.

 

Product Quantity Cat. No.  
Organelle Lights™ Mito-GFP1 kitO36210Order Now
Rhod-2, AM dye1 mgR1244Order Now

DEPARTMENTS

Buzzworthy

High-efficiency Transient Transduction of Human Embryonic Stem Cell–derived Neurons
 
High-efficiency transient transduction of human embryonic stem cell–derived neurons With baculoviral vectors.
Zeng J, Du J, Lin J et al. (2009) Mol Ther Epub ahead of print.

How can we transiently manipulate human embryonic stem cells to improve transplantation efficiency?
Human embryonic stem cells (hESCs) are an important source of cells for the generation of human neurons for basic research and therapeutic applications. Transplantation of hESC-derived neurons has the potential to repair damaged or diseased neurons, but efforts in this area have been limited by the poor survival and low functional recovery of grafted hESC-derived neurons. Genetic manipulation of hESC-derived cells prior to transplantation is one possible method of improving transplantation efficiency, but chromosomal integration of transgenes poses the risk of permanent expression of potentially harmful genes. Transient transduction is preferable for the expression of genes that are only beneficial during transplantation. To address this challenge, Zeng and colleagues developed a set of baculoviral vectors and demonstrate their use for successful transient transduction of hESC-derived neurons.

Methods
To develop a vector optimized for transient transduction of hESC-derived neurons, Zeng and colleagues investigated whether insect baculovirus–based vectors were suitable as a gene delivery system in these cells. They tested various eGFP expression cassettes in addition to the commercially available Organelle Lights™ ER-OFP and Mito-OFP baculoviral systems for transgene expression in hESC-derived neurons and in the brains of nude mice after transplantation.

Results
Baculoviral transduction of hESC-derived neurons was highly efficient (up to 80%), and transgene expression was detected as early as 1 day post-transduction. Transgene expression was stable for at least 1 month in cultured human neurons, and co-transduction of baculoviral vectors with Organelle Lights™ fluorescent protein constructs (using a modified protocol) resulted in successful targeting of the fluorescent proteins concurrent with eGFP transgene expression. Finally, baculovirus-modified hESC-derived neurons were transplanted into the brains of nude mice, where transgene expression was detected for up to 4 weeks after injection. Baculoviral transduction did not appear to inhibit neuronal function, and there was no apparent immune response to the transplanted cells, suggesting that baculoviral vectors are promising tools for the development of more efficient neural transplantation systems.

View bibliography reference

Free Online Technical Webinars Free online technical webinars
You are invited to join us for a series of biweekly technical webinars from the comfort of your desk. The webinars will initially focus on imaging-related applications, but we welcome your feedback for additional topics throughout the course of the year. Upcoming topics will be announced each month via email.

Presentations will last approximately 45 minutes, followed by 15 minutes for live Q&A.
Webinars Date Time
Fluorescence Imaging in Cell Biology with Updates August 18, 2009 10:00 a.m. PT
New Product Overview, part 1 September 1, 2009 10:00 a.m. PT


Missed our previous webinars? Find our recorded webinars here!





Live cell imaging of cell cycle and division
Live-cell imaging of cell cycle and division. U2OS cells were transduced with Premo™ FUCCI Cell Cycle Sensor, then imaged the next day using a 40x objective and standard FITC/TRITC filter sets. Time lapse imaging was performed on the DeltaVision® Core microscope. Images were collected every 15 min for 18 hr. 

Watch the Video!

Epitope Tag Antibodies

Over the years, a number of different epitope tags have been made available to researchers studying protein expression. The tags are recognized by antibodies—which can in turn be visualized by standard secondary detection techniques—and have proven useful for detecting proteins of interest via western blotting, ELISA, immunostaining, and immunoprecipitation. Invitrogen offers antibodies against several of the most popular epitope tags, including V5, Xpress, myc, histidine (his), and hemagglutinin (HA). Each antibody is also available in several conjugate forms and includes complete protocols for use.


Epitope tag antibodies 

Myc and V5 tags detected using epitope tag antibodies.
(A) Myc tag detected with anti-myc antibody. (B) V5 tag detected with anti-V5 antibody.


Product
Quantity Cat. No.
Anti-V5 Antibody50 µLR96025Order Now
Anti-Xpress™ Antibody50 µLR91025Order Now
Anti-myc Antibody50 µLR95025Order Now
Anti-histidine (c-terminal) Antibody50 µLR93025Order Now
Anti-HA Antibody100 µg326700Order Now
    


Environmental Sustainability Programs
Invitrogen, part of Life Technologies, is dedicated to protecting the environment. We have ongoing environmental sustainability programs that deliver products to reduce your environmental footprint, while helping you achieve your research goals.





IDC Latex Microspheres New Web Resource for IDC Latex Microspheres

Invitrogen offers a wide selection of UltraClean™ surfactant-free latex microspheres—also referred to as latex beads—for research and commercial applications. We have recently redesigned our IDC latex microspheres web page to include more information about the different surface modifications, detailed background information on the physical properties of microspheres, and a guide to selecting the best microsphere for your application. Also included are protocols for attaching latex microspheres to proteins or other biomolecules.




Post-Sorting Analysis Enabled by Low Toxicity of Cell Cycle Stain

Vybrant® DyeCycle™ Ruby Stain is used for cell cycle analysis in live cells by flow cytometry using the common 635 nm red laser for excitation. In collaboration with Derek Davies and Tina Luke from the FACS Laboratory at London Research Institute-Cancer Research UK, Invitrogen scientists have shown that Vybrant® DyeCycle™ Ruby Stain has low toxicity in live cells. This means that cells stained with Vybrant® DyeCycle™ Ruby Stain can be sorted on a flow cytometer into different phases of the cell cycle and then re-grown for subsequent analyses.  


Cell Viability
Post-sorting analysis of cell number and viability. HL-60 cells (5 x 106) were suspended in 10 mL of medium, 10 µL Vybrant® DyeCycle™ Ruby Stain was added, and the mixture was incubated for 15 min at 37°C. The cells were pelleted and resuspended in 1 mL PBS with 5 µL DAPI (200 µg/mL). (A) Live gating on DAPI negative cells followed by singlet gating based on area and height and sorted G0/G1 population (675/20 blue laser excitation and 660/20 red laser excitation). Mod FIT LT software was used to model the cell cycle, and the G0/G1 cells were then re-grown and evaluated for cell count (B) and viability (C).

Product Quantity Cat. No.  
Vybrant® DyeCycle™ Ruby Stain400 assaysV10273Order Now
Vybrant® DyeCycle™ Ruby Stain100 assaysV10309Order Now
    


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