In This Issue

FEATURED NEW PRODUCTS

 
Obtain Detailed Cell Cycle Kinetics — Click-iT® EdU HCS Assays

 New Modification-Specific Antibodies for the AKT Signaling Pathway — ABfinity™ Recombinant Monoclonal Antibodies

New Products for Cell &Tissue Analysis
 
Find Antibodies From Invitrogen with our new selection tools


NEW APPLICATIONS


 
Real-Time Visualization of Neuronal Growth Cone Dynamics Using BacMam 2.0
DEPARTMENTS


Check out the latest issue of BioProbes

BioProbes 62 
FEATURED ARTICLE:

 

More Information

FEATURED NEW PRODUCTS

what it is
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
how it works
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.

Labeling-Cell-Proliferation

High-content imaging and analysis of dual pulse labeling of cell proliferation.


Read More +

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®).
Quantity Cat. No.  
2-plate sizeC10350
10-plate sizeC10351
2-plate sizeC10352
10-plate sizeC10353
2-plate sizeC10354
10-plate sizeC10355
2-plate sizeC10356
10-plate sizeC10357
what they are
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

how they work
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.
 
ABfinity recombinant monoclonal
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.
Product Quantity Cat. No.  
ErbB-2 [pY1248] ABfinity™ Recombinant Rabbit Monoclonal Antibody100 μg700635Order Now
PLC-gamma [pY783] ABfinity™ Recombinant Rabbit Monoclonal Antibody100 μg700044Order Now
c-Met [pY1230⁄34⁄35] ABfinity™ Recombinant Rabbit Monoclonal Antibody10 mini-blots700139Order Now

 

NEW APPLICATIONS

BacMam Reagents
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.

BacMamLive-cell imaging of neuronal growth cone dynamics in cultured rat hippocampal neurons. Hippocampal tissue from rat postnatal day 3 animals was harvested and dissociated by gentle trituration in neural culture medium prior to plating onto glial feeder cultures, which were established a week prior on 35 mm glass-bottomed dishes (MatTek®, Ashland, MA). Undissociated hippocampal tissue was allowed to settle, and the supernatant containing neurons was transferred to a fresh vial. Cell density was determined with the Countess® Automated Cell Counter, and cells were resuspended at 50,000 cells/mL in complete neural culture medium plus mitotic inhibitors. The medium was removed from the glial feeder cultures and replaced with 2 mL of the neural cell suspension. 50 µL each of BacMam 2.0 GFP transduction control and CellLight™ MAP4-RFP were added to the dishes, and cells were placed into the cell culture incubator. A complete medium change was performed the following day, and cultures were allowed to mature for 2 weeks before imaging on a DeltaVison® DV Core (Applied Precision, Issaquah, WA), with partial media changes twice a week. GFP fluorescence was observed in neurons after 48 hr in culture. Images in the time-lapse series were collected with standard FITC and TRITC filter sets at 10-second intervals with 50-millisecond exposures. Images are a deconvoluted set of 10 projected optical sections 0.2 µm in depth.
Product Quantity Cat. No.  
BacMam GFP Transduction Control *BacMam 2.0*1 mLB10383Order Now
CellLight™ MAP4-RFP *BacMam 2.0*1 mLC10599 Order Now

 

DEPARTMENTS

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

 

Product Quantity Cat. No.  
Qdot® 655 Streptavidin Conjugate200 μLQ10121MPOrder Now

 

Phagocytosis in live mmm cells
  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.

 

Product Quantity Cat. No.  
MitoTracker® Red CMXRos20 x 50 µgM7512Order Now
HCS LipidTOX™ Green Neutral Lipid Stain1 eachH34475Order Now
ProLong® Gold Antifade Reagent5 x 2 mLP36934Order Now
Dulbecco’s Modified Eagle Medium, High Glucose1,000 mL11995040Order Now


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.

 

Apoptotic Cell
 
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.

 

Product Quantity Cat. No.  
Annexin V, Alexa Fluor® 488 conjugate
500 µLA13201Order Now
Annexin V, Alexa Fluor® 647 conjugate 500 µLA23204Order Now
Violet Ratiometric Membrane Asymmetry Probe⁄Dead Cell Apoptosis Kit 1 kitA35137Order Now
MitoProbe™ JC-1 Assay Kit 1 kitM34152Order Now
MitoProbe™ DiIC1(5) Assay Kit 1 kitM34151Order Now
Vybrant® FAM Caspase-3 and -7 Assay Kit 1 kitV35118 Order Now
YO-PRO®-1 Iodide 1 mLY3603Order Now
Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit1 kitV13241 Order Now

 

 Cell and Tissue Analysis Scientific Posters
The new Cell and Tissue Analysis posters page is a great resource for any lab. You can download any of our posters in PDF format and then print, save, or email it to a colleague.

Features:
 100+ scientific posters
 Searchable by research area
 Interactive Flash application
 Constant updates



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