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In This Issue
Obtain Detailed Cell Cycle Kinetics — Click-iT® EdU HCS Assays
||New Modification-Specific Antibodies for the AKT Signaling Pathway — ABfinity™ Recombinant Monoclonal Antibodies
Find Antibodies From Invitrogen with our new selection tools
Real-Time Visualization of Neuronal Growth Cone Dynamics Using BacMam 2.0
Buzzworthy — Tracking the Position and Orientation of a Single Virus
The View — Visualizing Adipogenesis in 3T3-L1 Cells
Proven Performers — Apoptosis Assays for Flow Cytometry
On the Web — Cell and Tissue Analysis Scientific Posters
Check out the latest issue of BioProbes
FEATURED NEW PRODUCTS
The Click-iT® EdU HCS Assay is a superior alternative to traditional antibody-based methods for detecting and quantitating cell proliferation for high-content screening (HCS) applications. The streamlined Click-iT® EdU HCS assays have fewer steps and require less time to detect newly synthesized DNA compared to traditional BrdU methods.
what it offers
- Simple method
- Highly specific detection
- Compatibility with any HCS platform
EdU is detected using a click reaction—a copper(I)-catalyzed reaction between an azide and an alkyne. In the Click-iT® EdU assay, the EdU nucleoside contains the alkyne, and the fluorescent detection reagent contains the azide. Using sequential pulses of the thymidine analogs EdU and BrdU, dual pulse labeling can be used to further define cell cycle kinetics.
- Learn More about Click-iT® EdU HCS Assays
High-content imaging and analysis of dual pulse labeling of cell proliferation.
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The effect of various drugs on DNA replication was assessed using dual pulse labeling. U2OS cells were pulsed with 10 µM EdU from the Click-iT® EdU Alexa Fluor® 488 Kit for 60 min, washed, then treated with chloroquine, rosiglitazone, acetaminophen, or etoposide, or left untreated for 23 hr. Cells were pulsed with 10 µM BrdU for 60 min, then fixed and denatured before performing the Click-iT® reaction to detect EdU (green). BrdU incorporation was detected using Anti-BrdU (clone MoBU-1)–Alexa Fluor® 594 Conjugate (red). Nuclei were counterstained with HCS NuclearMask™ Blue Stain. (A) Control cells dual-labeled with EdU and BrdU, imaged with a Zeiss Axiovert® 200M microscope. (B) DNA replication was decreased by several drugs, as indicated by a decrease in BrdU-positive cells compared to EdU-positive cells. Data were acquired using the ArrayScan® VTI platform (Thermo Scientific Cellomics®).
ABfinity™ antibodies are now available for phospho-ErbB2 (Y1248), phospho–PLC-gamma, (Y783) and phospho–c-Met (Y1230/34/35). These antibodies have been validated in applications such as western blotting, ELISA, and flow cytometry.
what they offer
- High sensitivity and specificity
- Unmatched lot-to-lot consistency, thanks to recombinant technology
- Extensive validation and characterization for multiple applications
ABfinity™ antibodies are generated by cloning the specific antibody genes and expressing 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.
- Learn More about ABfinity™ Recombinant Monoclonal Antibodies
|Phosphorylation site–specific ABfinity™ Recombinant Monoclonal Antibodies used in western blotting (A) and flow cytometry (B).
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(A) 3T3 cell lysates were untreated (lane 1) or treated with 50 ng/mL of PDGF for 30 min (lanes 2–4). Rabbit anti–PLC-gamma [pY783] antibody was preincubated with the nonphosphopeptide (lane 3) or the phosphopeptide (lane 4) before detection of PLC-gamma [pY783] in treated lysate. (B) Jurkat cells were fixed and permeabilized, then stained with 3 μg/mL of anti–PLC-gamma [pY783] followed by Alexa Fluor® 488 goat anti–rabbit IgG (black line). The gray line represents cells stained with Alexa Fluor® 488 goat anti–rabbit IgG alone. Competition was performed by preincubating anti–PLC-gamma [pY783] for 30 min in the presence of 50 µg/mL of the specific phosphopeptide (red line) or the nonphosphopeptide (blue line) to show specificity for the phosphopeptide only.
|ErbB-2 [pY1248] ABfinity™ Recombinant Rabbit Monoclonal Antibody||100 μg||700635|
|PLC-gamma [pY783] ABfinity™ Recombinant Rabbit Monoclonal Antibody||100 μg||700044|
|c-Met [pY1230⁄34⁄35] ABfinity™ Recombinant Rabbit Monoclonal Antibody||10 mini-blots||700139|
BacMam reagents use a modified baculovirus as a vehicle to efficiently deliver and express genes in mammalian cells. These ready-to-use fluorescent protein–based reagents contain targeting sequences fused to either GFP or RFP. Unlike expression vectors, BacMam reagents enable titratable and reproducible expression and offer high cotransduction efficiency, enabling multiple BacMam reagents to be used in the same cell. Recent improvements made to the BacMam system (BacMam 2.0) enable efficient transduction in a wider variety of cells, including primary neurons and stem cells, following a simple, one-step process.
|BacMam GFP Transduction Control *BacMam 2.0*||1 mL||B10383|
|CellLight™ MAP4-RFP *BacMam 2.0*||1 mL||C10599|
High-speed nanoscopic tracking of the position and orientation of a single virus
Kukura P, Ewers H, Muller C et al. (2009) High-speed nanoscopic tracking of the position and orientation of a single virus. Nat Methods 6(12):923–927.
Investigating individual molecules in biological systems requires seeing beyond the diffraction limit and down to the nanometer level. Traditional fluorescence-based probes are not useful for these types of experiments because they are subject to photobleaching and fluorescence saturation, and can’t be used to determine the orientation of the molecule under study. Kukura and colleagues circumvented these problems by developing a method that uses scattering interferometry and single-molecule fluorescence microscopy to deliver data on both the position and orientation of a single SV40 virus particle. Key to the success of this method was the use of a Qdot® nanocrystal label. When the label was applied to supported lipid bilayers, the team was able to observe single Qdot® nanocrystal–labeled virion particles sliding, tumbling, and rocking between nanoscopic regions separated by 9 nm. They postulate that similar approaches will be essential in studies of protein diffusion and nanomotion.
View the bibliography reference
- Learn More about Qdot® Nanocrystals
|Qdot® 655 Streptavidin Conjugate||200 μL||Q10121MP|
|| Adipogenesis visualized with LipidTOX™ Green Neutral Lipid Stain.
3T3-L1 cells were grown in DMEM + 10% FCS until 70% confluent. Cells were plated on coverslips, and adipogenesis was induced using the MDI method (1 µM dexamethasone, 0.5 mM IBMX, 10 µg/mL insulin in complete medium). Cells were grown for 10 days, with medium changes every 2 days. Cells were then labeled with 200 nM MitoTracker® Red CMXRos in complete medium for 30 min at 37ºC in the dark. Cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100. Cells were labeled with 1:1,000 HCS LipidTOX™ Green Neutral Lipid Stain in the dark for 30 min, then washed once with water and mounted with ProLong® Gold Antifade Reagent. Slides were cured for 24 hr in the dark, and imaging was performed on a Nikon A1R Point Scanning Confocal Microscope (60x; channels: FITC, Texas Red®, and direct light). Image contributed by Kevin Chambers, Life Technologies Corporation.
|MitoTracker® Red CMXRos||20 x 50 µg||M7512|
|HCS LipidTOX™ Green Neutral Lipid Stain||1 each||H34475|
|ProLong® Gold Antifade Reagent||5 x 2 mL||P36934|
|Dulbecco’s Modified Eagle Medium, High Glucose||1,000 mL||11995040|
Apoptosis—a fresh look at a dying science
Because it can be used to analyze a large number of cells in a single experiment, flow cytometry has been a method of choice for analyzing apoptosis within cell populations. It’s now even easier to find the assays you need for accurate, sensitive, and reliable analysis of apoptotic cells by flow cytometry. From classic Molecular Probes® Alexa Fluor®–annexin V conjugates to the newly released Violet Ratiometric Membrane Asymmetry Kit, you’ll be able to find what you need at our new Apoptosis Assays for Flow Cytometry page.
- Learn More about Apoptosis Assays for Flow Cytometry
Resolution of live and apoptotic cell populations. Jurkat cells (T cell leukemia, human) were treated with 10 μM camptothecin for 4 hr. Cells were stained with F2N12S from the Violet Ratiometric Membrane Asymmetry Kit according to the protocol. Samples were analyzed on a flow cytometer with 405 nm excitation using 585 nm and 530 nm bandpass filters. Live cells can be discriminated from apoptotic and dead cells by the relative intensities of the two emission bands from F2N12S. A = apoptotic cells, L = live cells, D = dead cells.
|Annexin V, Alexa Fluor® 488 conjugate
|Annexin V, Alexa Fluor® 647 conjugate||500 µL||A23204|
|Violet Ratiometric Membrane Asymmetry Probe⁄Dead Cell Apoptosis Kit||1 kit||A35137|
|MitoProbe™ JC-1 Assay Kit||1 kit||M34152|
|MitoProbe™ DiIC1(5) Assay Kit||1 kit||M34151|
|Vybrant® FAM Caspase-3 and -7 Assay Kit||1 kit||V35118|
|YO-PRO®-1 Iodide||1 mL||Y3603|
|Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit||1 kit||V13241|
Molecular Probes® The Handbook
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