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In This Issue
FEATURED NEW PRODUCTS
NEW APPLICATIONS
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DEPARTMENTS
Check out the latest issue of BioProbes
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FEATURED NEW PRODUCTS
Click-iT® EdU HCS Assays
| 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
| 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.
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ABfinity™ Recombinant Monoclonal Antibodies
| 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
| 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.
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| Product | Quantity | Cat. No. | |
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| 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 | |
NEW APPLICATIONS
Real-Time Visualization of Neuronal Growth Cone Dynamics Using BacMam 2.0
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.
Live-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.
DEPARTMENTS
Buzzworthy
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
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| 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. |
| Product | Quantity | Cat. No. | |
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| 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. |
| Product | Quantity | Cat. No. | |
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| Annexin V, Alexa Fluor® 488 conjugate | 500 µL | A13201 | |
| 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 | |
 | 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:
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