ProbesOnline November 2009

In This Issue


New AKT Pathway Antibodies ABfinity™ Recombinant Monoclonal Antibodies

Take Charge of Your Cell Counting Countess™ Automated Cell Counter New Features

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


Imaging Viral RNA Synthesis Click-iT® RNA Assays


what they are
The PI3K/AKT signaling pathway is central to major cell functions such as cell growth, survival, apoptosis, angiogenesis, and the cell cycle. We now offer highly specific ABfinity™ recombinant rabbit monoclonal antibodies to AKT and other pathway members.

what they offer

  • High sensitivity and specificity
  • Unmatched lot-to-lot consistency, thanks to recombinant technology
  • Extensive validation and characterization for multiple applications

how they work
ABfinity™ antibodies are generated by cloning the specific antibody genes and producing them in a mammalian expression system, ensuring maximal lot-to-lot consistency. Total and phosphorylation site–specific antibodies are available for the study of the AKT signaling pathway and its role in glucose metabolism, cell cycle, cell survival, cell adhesion, and angiogenesis.

 ABfinity™ rabbit momoclonal antibody

Immunocytochemistry analysis of mouse fibroblast cells. (A) Mouse fibroblast cells were treated with (right) or without (left) 10 µg/mL insulin and labeled with AKT [pS473] ABfinity™ Recombinant Rabbit Monoclonal Antibody. (B) For the insulin-treated cells in (A), the signal is knocked down after incubation with the phosphopeptide used as an immunogen (left) but not with the nonphosphopeptide (right). Alexa Fluor® 488 goat anti–rabbit IgG at 1:1,000 was used as the secondary antibody. Nuclei were stained with one of the Hoechst dyes.

ABfinity™ Rabbit Monoclonal Antibodies

Quantity Cat. No. Reactivity: validated (expected) Applications
AKT [pS473] 100 μg 700392 Hu, Ms (B, Ch, Cn, Cp, Eq, Mk, Rt, X, Z) E, F, IF, IHC, W Order Now
PKC-θ [pT538] 100 μg 700043 Hu (B, Cp, Ms, Rt, X) F, IF/ICC, IHC, WB Order Now
Mnk1 [pT197/pT202] 100 μg 700242 Hu (B, Ch, Cn, Cp, Eq, Mk, Ms, P, Rt, Sw, X, Z) F, IF/ICC, WB
Order Now
Cul-2 100 μg 700179 Hu, Ms, Rt (B, Cn, Cp, Eq, Mk, Or, X) F, IF/ICC, IHC, WB Order Now
IRAK4 100 μg 700026 Hu (B, Cn, Eq, Qu, Rt, Sh, Sw) F, IF/ICC Order Now
AMPKβ1 [pS182] 100 μg 700241 Hu (B, Ch, Cn, Eq, Ms, Or, Rt, X) F, IF/ICC, IHC, WB Order Now
Smad1/5 [pS463/pS465] 100 μg 700047 Hu (B, Ch, Cn, Cp, Eq, Mk, Ms, Rt, Sh, Su, Sw, X, Z) F, IF/ICC, IHC Order Now
4E-BP1 [pT37] 100 μg 700238 Hu (B, Eq, Hu, Ms, Rt, Z) E, F, IF/ICC, IHC, WB Order Now
STAT4 100 μg 700185 Hu, Ms, Rt (Eq, Sw) E, F, IF/ICC, IHC, WB Order Now
SUMO-3 100 μg 700186 Hu, Ms, Rt (Cp, Mk) F, IF/ICC, IHC, WB Order Now
CASP3 [D175] (clone 9H19L2) 100 μg 700182 Hu (B, Cn, Cp, Eq, Fe, Ha, Ms, P, Rb, Rt, Sh, Sw, X) E, F, IF/ICC, IHC, WB Order Now
Reactivity: B, bovine; Ch, chicken; Cn, canine; Cp, chimpanzee; Eq, equine; Fe, feline; Gf, goldfish; Ha, hamster; Hu, human; Ma, mammalian; Mk, monkey (rhesus); Ne, nematode; Or, orangutan; P, primate; Qu, quail; Rb, rabbit; Rt, rat; Sh, sheep; Sw, swine; X, Xenopus; Z, zebrafish.
E, ELISA; F, flow cytometry; ICC, immunocytochemistry; IF, immunofluorescence; IHC, immunohistochemistry; IP, immunoprecipitation; WB, western blotting
what it is
The Countess™ Automated Cell Counter has recently released features that provide even greater accuracy and cell-counting control both before and after data are generated. The new features include viability validation, cell gating, and a personal protocol maker. In addition, you can now purchase an extended warranty to add coverage to your existing one-year manufacturer’s warranty.

what it offers

  • Verify that cells are being properly counted directly from the screen
  • Further analyze cell samples and easily manipulate data immediately after a cell count
  • Reset differentiation based on cell size, and save experimental parameters for subsequent counts
how it works
The Countess™ Automated Cell Counter uses trypan blue staining combined with a sophisticated image analysis algorithm to produce accurate cell and viability counts in just 30 seconds. The algorithm also measures the average size of live, dead, and total cells, to give you all the data you need to proceed with your experiments. The new features come with a new instrument or can be downloaded for free to an existing instrument.


Countess™ Automated Cell Counter New Features.

  • Viability validation
  • Cell gating
  • Personalized protocol

Countess™ Automated Cell Counter Products

Product Cat. No. Quantity  
Countess™ Automated Cell Counter C10227 1 each Order Now
Countess™ Automated Cell Counter Starter Kit
(includes 1 cell counter and 11 boxes of slides)
C10310 1 kit Order Now
Countess™ Automated Cell Counter Lab Starter Kit
(includes 1 cell counter and 101 boxes of slides)
C10311 1 kit Order Now
One Year Extended Warranty* (At time of purchase or within one year of the purchase of the Countess™ Automated Cell Counter)
One Year Deferred Warranty* (More than 1 year after the purchase of the Countess™ Automated Cell Counter) 

* The extended warranty is only offered in the US and Canada.



Click-iT® RNA assays are ideal for imaging global RNA synthesis using traditional fluorescence microscopy or high-content screening (HCS). The assays employ an alkyne-modified nucleoside, EU (5-ethynyl uridine), that is fed to cells and incorporated into nascent RNA. The small size of the alkyne tag enables efficient incorporation by RNA polymerases without any apparent changes to the RNA levels of several housekeeping genes. Detection is accomplished using an Alexa Fluor® azide, and results are available in 30 minutes—following EU incubation, simply fix and permeabilize your sample, then perform the copper-catalyzed click reaction.

Little is understood about the sites of viral RNA synthesis, including the transport and fate of nascent RNA inside and outside the viral replication complex. The Click-iT® RNA Imaging and Click-iT® RNA HCS kits provide exciting new alternatives to antibody-based BrU or BrUTP assays for studying viral RNA synthesis in infected cells. Unlike BrUTP, EU does not appear to require a transfection reagent, and the small size of the Alexa Fluor® azide relative to the anti-BrdU antibody will enable facile detection of both ssRNA and dsRNA. Click-iT® RNA imaging assays provide the tools necessary to determine levels of viral RNA synthesis as well as the subcellular localization of RNA in host cells, which will aid in understanding viral gene expression and genome replication.

RNA Synthesis  

Subcellular localization of RNA in Vero cells.
Vero cells were pretreated with 2 μM actinomycin D to inhibit host cell transcription, then infected with Tacaribe virus. Infected cells were incubated with 2 mM EU for 1 hr, followed by cold methanol fixation and permeabilization with Triton® X-100. Nascent RNA (green) was detected with the Click-iT® RNA Alexa Fluor® 488 Imaging Kit. Viral nucleoprotein was detected with a directly labeled Alexa Fluor® 594 monoclonal antibody (red). Colocalization of EU and virus nucleoprotein indicates transcription sites in the host cells (yellow).

Click-iT® RNA Products

Product Cat. No. Quantity  
Click-iT® RNA Alexa Fluor® 488 HCS Assay, 2-plate size C10327 1 kit Order Now
Click-iT® RNA Alexa Fluor® 594 HCS Assay, 2-plate size C10328 1 kit Order Now
Click-iT® RNA Alexa Fluor® 488 Imaging Kit, for 25 coverslips C10329 1 kit Order Now
Click-iT® RNA Alexa Fluor® 594 Imaging Kit, for 25 coverslips C10330 1 kit Order Now
5-ethynyl uridine (EU) E10345 5 mg Order Now



The Click-iT® EdU Assay Sheds Light on the Role of Amyloid-β in Neural Disease and Neurogenesis

Differential processing of amyloid-β precursor protein directs human embryonic stem cell proliferation and differentiation into neuronal precursor cells.
Porayette P et al. (2009) J Biol Chem 284:23806–23817.

What mechanisms underlie the neurodegenerative effects of Alzheimer’s disease and Down syndrome?
Amyloid-β precursor protein (AβPP) is a transmembrane protein with important neural functions. It is processed in vivo by two distinct pathways, termed amyloidogenic and non-amyloidogenic; products of the latter processing pathway are important growth factors, while the former pathway yields a toxic protein known as amyloid-β (Aβ) that is implicated in Alzheimer’s disease, Down syndrome, and neural damage associated with head trauma.

Methods and Results
In their recent study, Porayette and colleagues investigated the role of AβPP products in the regulation of human embryonic stem cell (hESC) proliferation and differentiation. The authors report that Aβ protein is endogenously produced by hESCs; using the Click-iT® EdU Alexa Fluor® assay, they demonstrated that the addition of exogenous Aβ protein induced hESC proliferation. Inhibition of hESC secretase enzymes markedly suppressed stem cell proliferation, implicating these enzymes in the differential processing of AβPP.

The regulation of this differential processing mechanism may play a role in the neuropathology associated with Alzheimer’s disease, Down syndrome, and debilitating head injuries, opening a potential new avenue for therapeutic treatment.

View bibliography reference

  • Learn More about Click-iT® EdU Cell Proliferation Assays

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
Tools for iPSC Research
November 17, 2009 10:00 a.m. PT
Tools for iPSC Research November 24, 2009 10:00 a.m. PT

Missed our previous webinars? Find our recorded webinars here!

Calcium Microscopy

Monitoring synaptically evoked calcium transients by two-photon laser-scanning microscopy in intact brain tissue using fluorescent dyes.
A CA1 rat pyramidal neuron was microinjected with 300 μM fluo-5F, a calcium indicator (Kd 800 nM), and 20 μM Alexa Fluor® 594 hydrazide. Both dyes were excited at 800 nm using two-photon excitation. In the video, calcium transients are shown following individual evoked action potentials in presynaptic axons (evidenced by the appearance of a yellow-fluorescent signal against the Alexa Fluor® 594 dye–labeled red-fluorescent background). Using a ratio of the two signals, absolute calcium concentration in individual dendritic spines can be calculated. Such high-resolution optical imaging combined with electrophysiology allows hundreds of transmission events to be recorded at identified synapses, providing temporal and spatial precision that would have been unimaginable just a decade ago. Image courtesy of Thomas Oertner, FMI Institute for Biomedical Research, Basel, Switzerland, and Karel Svoboda, HHMI, Janelia Farms Research Institute, Virginia, US.

Watch the video NEW!

Proven Performers — Qdot® Nanocrystal Conjugates for Flow Cytometry

Fundamentally, Qdot® nanocrystals are fluorophores—substances that absorb photons of light, then reemit photons at a different wavelength. However, these particles fluoresce in a way that’s completely different from traditional fluorophores. Their intrinsic brightness is often many times greater than that observed for organic fluorophores, and their photostability is many orders of magnitude greater. When conjugated to molecules such as primary or secondary antibodies or streptavidin, Qdot® nanocrystals provide exceptional fluorescence with full biofunctionality for a wide variety of life science applications, including flow cytometry and live-cell and fixed-cell imaging.

Qdot® nanocrystal primary antibody conjugates are increasingly used in multispectral flow cytometry. The availability of more color options allows more efficient experiments by obtaining more information with less sample, in less time. The use of these conjugates allows the addition of one to six colors, all excited from the violet laser. They can be used in combination with existing organic dyes to increase the number of detectable parameters. We offer human and mouse antibodies for flow cytometry conjugated to Qdot® 605, Qdot® 655, Qdot® 705, and Qdot® 800 nanocrystals.

Antigen Expression Lymphocytes

Multiplexed analysis of antigen expression in human peripheral blood lymphocytes (PBLs). PBLs were labeled with anti-CD45 Qdot® 705, anti-CD4 Qdot® 605, anti-CD3 Qdot® 655, anti-CD8 APC-Alexa Fluor® 750, and anti-CD2 Fluorescein. Samples were run on a BD LSR II flow cytometer. Plots are gated on lymphocytes by side scatter. Axes are labeled with the filters used; plots are labeled with compensation values.

  Novel Recombinant Monoclonal Antibodies
Novel ABfinity™ recombinant monoclonal antibody technology ensures consistent results with every validated application, time after time. Learn more about the excellent results you can expect from ABfinity™ antibodies for your research at our web page dedicated to ABfinity™ technology.


Improved Qdot® Nanocrystals Web Page
The extraordinary properties of Qdot® nanocrystals and bioconjugates make them ideal for experiments requiring long-term photostability or single-excitation multicolor analysis, including flow cytometry and cellular imaging. With our improved web resource, it’s now easier to view all Qdot® product offerings and find out about new releases. You can also conveniently learn about Qdot® applications and download the latest publications by research area or application.


Now available! The all new Invitrogen Immunology Systems Catalog for 2010—find all your immunology and cell biology research tools in one place!

  • ELISAs and Luminex® assays
  • GIBCO® specialty media and growth factors
  • Cell culture and expansion
  • Dynal® cell isolation
  • siRNAs and TaqMan® Gene Expression Assays
  • Flow cytometry
  • Instrumentation
  • And much more!

The Immunology Systems Catalog is also a great benchtop resource for signaling pathways and key laboratory application protocols. To learn more and get your own copy, contact your local Invitrogen Account Manager or visit the Invitrogen website.

Request yours today!

Molecular Probes® The Handbook

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