ProbesOnline™ November 2013—New products for accurate RNA quantitation, simplified microscopy, and more…
In this issue
FEATURED NEW PRODUCTS
Simplified light microscopy—EVOS® XL Imaging System Accurately quantitate RNA—Quant-iT™ RNA HS Reagent ABfinity™ recombinant antibodies—New antibody for c-Kit (CD117) New antibodies for studying regulatory T cells by flow cytometry—Foxp3 antibody conjugates
Molecular Probes® webinar series
Free on-demand webinar—
Basic techniques in autophagy research
The American Society for Cell Biology® (ASCB®)
2013 Annual Meeting
The New Orleans Ernest N. Morial Convention Center
New Orleans, Louisiana, USA
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Life in the Lab magazine
Find product information, science news articles, special offers, and more
FEATURED NEW PRODUCTS
Simplified light microscopy
EVOS® XL Imaging System
What it is
Combining advanced ergonomic design, an on-board microprocessor, LED illumination, and a sensitive color camera, the EVOS® XL Imaging System is an ideal instrument for transmitted-light microscopy. The precision components and unique design functionality of the EVOS® XL Imaging System make it a perfect choice for colorimetric applications like H&E staining and immunohistochemistry.
What it offers
- Simplicity—intuitive user interface makes capturing images easy for all researchers
- Power—superb optics, including high-resolution objectives, for the most demanding transmitted-light applications
- Convenience—compact footprint for use on a lab bench or in a cell culture hood
How it works
The EVOS® XL Imaging System is a fully integrated imaging system that combines high-quality optics, a 15” high-resolution LCD display, and a digital 3 MP CMOS color camera. The on-board computer makes the EVOS® XL Imaging System easy to operate, and the intuitive interface facilitates image acquisition and analysis. Everything you need to acquire, manipulate, save, and transfer an image is built into the microscope.
With a 5-position objective turret and a 4-position condenser turret, the EVOS® XL Imaging System is ideal for a range of applications, including routine cell and tissue culture, cell confluence determination, stem cell growth and differentiation, developmental biology, and tissue slice analyses. And the compact footprint makes it as easy to use the EVOS® XL Imaging System in a cell culture hood as on the bench.
- Learn more about the EVOS® XL Imaging System ›
Quant-iT™ RNA HS Reagent
What it is
The Quant-iT™ RNA HS Reagent allows easy and accurate quantitation of RNA. Unlike our RiboGreen® reagent and RiboGreen® reagent–based kits, the Quant-iT™ RNA HS Reagent can be used to accurately measure RNA even in the presence of DNA and other common contaminants such as salts, free nucleotides, solvents, detergents, and protein. Additionally, the Quant-iT™ RNA HS Reagent is highly selective for larger RNA (e.g., mRNA) over microRNA (miRNA). The Quant-iT™ RNA HS Reagent is the same reagent included in our Quant-iT™ RNA Assay Kit and Qubit® RNA HS and BR Assay Kits, but is now available separately.
What it offers
- Sensitivity—accurately detects as little as 5 ng RNA, even in the presence of miRNA or DNA
- Accuracy—achieves more accurate results than with UV absorbance readings or with other fluorogenic RNA-detection reagents
How it works
On binding to RNA, the fluorescence of the Quant-iT™ RNA HS Reagent increases several hundred–fold, producing a very high signal-to-background ratio. High signal and low background mean exceedingly high sensitivity—up to 1,000 times more sensitive than UV absorbance readings. Depending on the concentration used in the assay, the Quant-iT™ RNA HS Reagent can measure with high confidence very low levels of RNA (25–500 ng/mL), or a more broad range (0.1–5 μg/mL), or can be optimized for your samples. The fluorescence can be detected on the Qubit® 2.0 Fluorometer or with a fluorescence microplate reader (the excitation/emission maxima of RNA-bound Quant-iT™ RNA HS Reagent are 644/673 nm).
- Learn more about Quant-iT™ technology
RNA selectivity and sensitivity of the Quant-iT™ RNA assay. Triplicate 10 µL samples of Escherichia coli rRNA (open triangle), DNA (open circle), or a 1:1 mixture of RNA and DNA (filled square) were assayed with the Quant-iT™ RNA Assay Kit. Excitation was at 630 nm. Fluorescence was measured at 680 nm and plotted versus the mass of nucleic acid for the RNA alone, the mass of nucleic acid for the DNA alone, or the mass of the RNA component in the 1:1 mixture. The coefficient of variation (CV) of replicate RNA determinations was ≤10%. The inset is an enlargement of the graph at the lower nucleic acid quantities to show the sensitivity of the assay. Background fluorescence has not been subtracted.
New antibody for c-Kit (CD117)
What they are
ABfinity™ recombinant monoclonal and oligoclonal antibodies offer consistent results, minimizing the need to revalidate working antibody dilutions for your experiments each time you order. Life Technologies currently offers hundreds of ABfinity™ recombinant antibodies, and we are actively developing more.
c-Kit (CD117) is a cell surface marker of hematopoietic stem cells that functions as a cytokine receptor, encoded by the KIT gene. Upon binding to stem cell factor (c-Kit ligand), it forms a dimer that activates tyrosine kinase activity and phosphorylation of key molecules in several pathways (e.g., PIK3R1, PLCG1, SH2B2/APS, and CBL) involved in cell survival, proliferation, and differentiation. c-Kit is also involved in hematopoietic development, with signaling through c-Kit resulting in mobilization of progenitor cells from the bone marrow into the bloodstream.
What they offer
- Specificity—undergo rigorous validation
- High performance—proven consistency from lot to lot
- Efficiency—detect low-level targets with a small sample
How they work
ABfinity™ antibodies are produced by transfecting mammalian cells with high-level expression vectors containing immunogen-specific rabbit antibody heavy and light chain cDNA. This highly reproducible process results in superb consistency in lot-to-lot antibody performance.
ABfinity™ oligoclonal antibodies are mixtures of recombinant monoclonal antibodies. These combine the improved signal strength that can come from using polyclonal antibodies, with the highly reproducible results you get from ABfinity™ monoclonal antibodies.
Detection of c-Kit in cell lysates on western blots. Western blot analysis was performed on whole cell lysates (30 μg each) from HeLa (lane 1), Jurkat (lane 2) and K562 (lane 3) cells. Endogenous c-Kit was detected at ~80 kDa using c-Kit ABfinity™ recombinant rabbit monoclonal antibody at 1 μg/mL. HRP-conjugated goat anti–rabbit IgG was used as the secondary antibody. The blot was developed using a chemiluminescence (ECL) method.
Foxp3 antibody conjugates
What are they
The anti–mouse Foxp3 antibody (clone 3G3) conjugates can be used for intracellular detection of mouse Foxp3, a central regulator of T cell activity. The Foxp3 monoclonal antibody is available conjugated to allophycocyanin (APC), phycoerythrin (PE), or fluorescein (FITC). Also available are the buffers required to facilitate intranuclear staining with Foxp3 antibody conjugates for flow cytometry applications.
What they offer
- Validation—fully validated in flow cytometry applications for specificity against mouse Foxp3
- Versatility—three fluorescent antibody conjugates to fit your research needs
How they work
Foxp3 is a transcription factor that is expressed constitutively at high levels in regulatory T cells (Tregs). In resting conventional T cells (CD4+ CD25–), Foxp3 expression is restricted and, following T cell receptor (TCR) activation, is expressed transiently. The growth factor TGF-beta induces expression of Foxp3 in naive T cells, driving their development into Foxp3+ Tregs. These “induced” or “adaptive” Tregs are phenotypically similar to the “natural” Tregs (CD4+ CD25high Foxp3+) that make up the majority of Treg cells in mice.
Because Foxp3 is an intracellular protein, Life Technologies offers prepackaged reagents (Foxp3 Transcription Factor Fixation and Permeabilization Buffer A and Buffer B) that are mixed together to make a working solution for fixation and permeabilization of cells prior to interrogation with anti-Foxp3 antibody.
- Learn more about Life Technologies antibodies for flow cytometry
Detection of Foxp3+ cells by flow cytometry. C57Bl/6 splenocytes were stained with FITC anti–mouse CD4, followed by intracellular staining with (A) 0.125 µg APC mouse IgG1 isotype control or (B) 0.125 µg APC anti–mouse Foxp3.
Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes
Super-resolution microscopy (SRM) refers to a group of techniques that significantly enhance the resolution of light microscopy . Traditional visible-light microscopy techniques, such as confocal or wide field, have a resolution limited by the diffraction limit of light. SRM modalities break this limit, thereby providing greater resolution. Of these SRM methodologies, one in particular—stochastic optical reconstruction microscopy (STORM)—stands out as especially attractive because of the excellent resolution provided (currently the limit is 10 nm) , and because widely available fluorophores can be used in STORM .
The laboratory of Dr. Xiaowei Zhuang, who originally described STORM , has expanded the number of fluorophores that can be used for STORM. Shim et al. employ commonly used organelle-specific dyes (LysoTracker® Red, MitoTracker® Red, and ER-Tracker™ Red) to perform STORM imaging of lysosomes, mitochondria, and endoplasmic reticulum (ER). Moreover, because those are live-cell probes, the demonstration of their use in STORM further extends the capacity for SRM imaging of live cells.
Two-color stochastic optical reconstruction microscopy (STORM) image of microtubules (green) and mitochondria (red). BS-C-1 cells were aldehyde-fixed, reduced with sodium borohydride, and labeled with primary antibodies to tubulin and the mitochondrial marker Tom20. Secondary antibodies conjugated with the photoswitchable dye pairs Alexa Fluor® 405/Alexa Fluor® 647 and Cy®3/Alexa Fluor® 647 were used to label tubulin and Tom20, respectively. Image courtesy of Xiaowei Zhuang, Harvard University.
Fluorophores for stochastic optical reconstruction microscopy (STORM).
Make ordinary tasks extraordinary
Simple instrumentation for your cell culture room
Quantitative (determining cell counts) or qualitative (examining cell morphology) analysis of cells in the cell culture room can be laborious and time-consuming. Benchtop instruments from Life Technologies can help you generate extraordinary results from these ordinary tasks. Whether you need to analyze fluorescent or nonfluorescent cells, these simple yet affordable instruments all have small footprints to fit right inside your hood or next to the incubator.
Benchtop instruments for the cell culture room.
|Fluorescent cell samples||Nonfluorescent cell samples|
EVOS® FLoid® Cell Imaging Station
EVOS® XL Core Imaging System
Tali® Image-Based Cytometer
Countess® Automated Cell Counter
- Learn more about instruments for the cell culture room
|Benchtop instruments for your culture room. Left to right: EVOS® FLoid® Cell Imaging Station, EVOS® XL Core Imaging System, Tali® Image-Based Cytometer, Countess® Automated Cell Counter.|
Newly revised mobile apps—Quickly find what you need for flow cytometry or imaging applications
The newly revised Molecular Probes® Flow Cytometry and Molecular Probes® Fluorescence Imaging mobile apps† are designed to help you find fluorescent reagents, kits, and protocols for cell biology applications. Now you can use your mobile device to:
The mobile apps also include fluorescence excitation and emission data for each product, making experimental design and setup easier than ever. Listed below are the application areas and product types included in both apps.
|Application areas||Product types|
Growth of Escherichia coli flagella imaged by fluorescence microscopyFlagellar filaments of E. coli strain HCB1737 were labeled with thiol-reactive Alexa Fluor® 488 C5 maleimide (green), cultured for an additional period of time, and then labeled with Alexa Fluor® 546 C5 maleimide (red). Figure provided by Howard C. Berg, Alan S. Stern, and Lynda Turner, Harvard University, Cambridge, Massachusetts, and reproduced with permission. A similar figure and more information can be found in Turner L, Stern AS, Berg HC (2012) Growth of flagellar filaments of Escherichia coli is independent of filament length. J Bacteriol 194(10):2437–2442.
Multiplex western blotting with fluorescent antibody probes: Long-wavelength Qdot® and Alexa Fluor® conjugates for sensitive and quantitative detection
In the Protein Expression section of BioProbes 69, the article “Multiplex western blotting with fluorescent antibody probes” describes the use of long-wavelength Qdot® and Alexa Fluor® secondary antibody conjugates for sensitive western blot detection. With recent advances in Qdot® probe technology, near-IR–emitting fluorescent dyes, and fluorescence imaging platforms, researchers are now able to obtain detection sensitivities from fluorescence-based methods that rival those obtained from enhanced chemiluminescence (ECL). Importantly, probes with long-wavelength (red to near-IR) emission allow signal collection beyond the typical autofluorescence emitted by standard nitrocellulose and PVDF membranes, resulting in low background fluorescence and superior signal-to-noise ratios. Furthermore, unlike ECL techniques, fluorescence-based detection methods provide the capability to detect and quantitate multiple antigens on a single blot without stripping and reprobing.
Learn more about our long-wavelength Alexa Fluor® and Qdot® (WesternDot™) secondary antibody conjugates, including:
- Alexa Fluor® 680 and Alexa Fluor® 790 anti–mouse IgG and anti–rabbit IgG antibody conjugates
- WesternDot™ 585 anti–mouse IgG, anti–rabbit, and anti–goat IgG antibody conjugates
- WesternDot™ 625 anti–mouse IgG, anti–rabbit, and anti–goat IgG antibody conjugates
- WesternDot™ 655 anti–mouse IgG, anti–rabbit, and anti–goat IgG antibody conjugates
- WesternDot™ 800 anti–mouse IgG and anti–rabbit IgG antibody conjugates
Comparison of western blot protein detection based on enhanced chemiluminescence (ECL) and Qdot® fluorescence. Serial dilutions of GAPDH (quantities of 3–750 ng protein) were run on NuPAGE® Novex® 4–12% Bis-Tris precast gels and transferred to iBlot® nitrocellulose membranes using the iBlot® Gel Transfer Device. The membranes were then probed with mouse anti-GAPDH antibodies followed by either ECL detection using a horseradish peroxidase goat anti–mouse IgG conjugate, or fluorescence detection using Qdot® 655 goat anti–mouse IgG conjugate. Images were collected using the Fujifilm® LAS-4000 gel imager. Partial blot images (insets) show GAPDH detection, and the graph shows the detection sensitivities of the two techniques. AU: arbitrary units.
§ What's new with the BioProbes® Journal?
We are bringing our award-winning BioProbes® articles to you sooner. We will be publishing new BioProbes® articles online every month and highlighting those articles here. That way, we can keep you up-to-date on new fluorescence technologies and cell biology applications. Check back frequently and watch BioProbes 69 take shape!
Molecular Probes® webinar series
Free on-demand webinar—Basic techniques in autophagy research
Autophagy is vital for maintaining normal cell function and enables cells to survive environmental stresses, including nutrient deprivation and physiological responses to pathogens or disease. Disturbances in autophagy are associated with metabolic and neurological diseases as well as cancer.
This on-demand webinar, “Basic techniques in autophagy research”, introduces a series of analytical tools and techniques to help identify and interrogate key features of autophagy. Topics include:
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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