ProbesOnline™ Newsletter

In this issue

FEATURED NEW PRODUCTS 

EVOS® XL Core Cell Imaging System Simplified cell imaging right in the hood—EVOS® XL Core Cell Imaging System
   
anti-mCherry antibody Expanding our anti–fluorescent protein antibody offerings—introducing the new anti-mCherry antibody
   
ABfinity™ recombinant antibodies ABfinity™ recombinant antibodies—new antibodies for Src
   
MyQubit Amplex® Red Cholesterol Assay Accurate cholesterol quantitation—MyQubit Amplex® Red Cholesterol Assay
   
CellROX® Flow Cytometry Assay Kits Detect reactive oxygen species (ROS) in live cells using flow cytometry—CellROX® Flow Cytometry Assay Kits
   
anti–mouse CD conjugates for flow cytometry Antibody conjugates for the detection of mouse cell surface markers—new anti–mouse CD conjugates for flow cytometry

NEW APPLICATIONS

New applicationsA patient-derived stem cell model of hereditary spastic paraplegia with SPAST mutations

 

PROVEN PERFORMERS

Cell proliferation A suite of tools for measuring cell proliferation

 UPCOMING EVENTS

Flow cytometry apoptosis workshop
NEW DATE: November 5-7, 2013
The Life Technologies Customer Experience Center
Frederick, MD

OTHER PUBLICATIONS

BioProbes® Journal of Cell Biology Applications
BioProbes 69  
 
The Molecular Probes® Handbook
Molecular Probes Handbook
 

FEATURED NEW PRODUCTS

Simplified cell imaging right in the hood—EVOS® XL Core Cell Imaging System

What it is
The EVOS® XL Core Cell Imaging System is a digital, transmitted-light, inverted-imaging system for cell and tissue culture applications and routine cell maintenance. Its color camera and high-quality optical system deliver high-definition images with exceptional ease. With a small footprint and an ergonomic design, the all-in-one EVOS® XL Core Cell Imaging System is a perfect addition to any cell culture room or facility.


What it offers

  • Easy installation; no maintenance, assembly, alignment, or calibration
  • Configured for four objectives
  • Fits and operates inside a cell culture hood
  • All-in-one design: digital camera, precision optics, LCD display, and USB-based storage

How it works
The EVOS® XL Core Cell Imaging System is an integrated transmitted-light, inverted-imaging system that combines high-quality optics, a 12.1-inch high-resolution LCD display, and a digital color camera. The intuitive mouse-based user interface and the integrated software make it simple to acquire images. The software provides a variety of features, including color temperature control. You will be astonished at how easy it is to operate, and amazed at how extraordinarily good your images look on-screen. The compact footprint makes it easy to use the EVOS® XL Core Cell Imaging System wherever needed; the entire system can be easily moved into a cell culture hood.

 

EVOS® XL Core Cell Imaging System   The EVOS® XL Core Cell Imaging System.  
Product Quantity Cat. No.
EVOS® XL Core Cell Imaging System
1 eachAMEX1000



Expanding our anti–fluorescent protein antibody offerings—introducing the new anti-mCherry antibody

What it is
Because of its improved brightness, superior photostability, and extremely rapid maturation rate, the mCherry monomeric red fluorescent protein is becoming the red fluorescent protein of choice for monitoring physiological processes and detecting transgenic expression. To expand the options for researchers using the mCherry red fluorescent protein, we now provide anti-mCherry rat monoclonal antibody.



What it offers

This anti-mCherry antibody performs well in the following applications:

  • Western blot analysis
  • Immunocytochemistry (including flow cytometry)
  • Immunoprecipitation


How it works

To generate this antibody, the full-length mCherry protein was used as the immunogen. The resulting affinity-purified rat monoclonal IgG2a antibody has been demonstrated to detect native and denatured forms of mCherry or mCherry fusion proteins in western analysis and immunocytochemistry applications. No cross-reactivity was demonstrated against several forms of GFP and RFP in western analysis, immunocytochemistry imaging, and flow cytometry. This antibody has also been validated in immunoprecipitation applications.

 

anti-mCherry antibody Utility of the anti-mCherry antibody for immunocytochemistry imaging and flow cytometry. (A) U2OS cells expressing mCherry, denatured and probed with mCherry rat monoclonal antibody and Alexa Fluor® 647 goat anti–rat IgG. (B) Selectivity of the anti-mCherry antibody was demonstrated with flow cytometry using U2OS cells expressing plasma membrane–targeted GFP (light green line), emerald GFP (dark green line), plasma membrane–targeted TagRFP (purple line), P62-mKate2 (red line), or mCherry (pink line), probed with anti-mCherry that had been conjugated to Pacific Blue™ dye.

Product Quantity Cat. No.
mCherry Rat Monoclonal Antibody1 vialM11217



ABfinity™ recombinant antibodies—new antibodies for Src

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.

Src is a non-receptor tyrosine kinase that associates with the cytoplasmic region of various cell-surface receptors to facilitate signal transduction. Src is involved in cytoskeletal organization, cell cycle regulation, and apoptosis. In humans, the gene encoding Src is present on chromosome 20.



What they offer

  • Specificity—undergo rigorous validation
  • High performance—proven consistency from lot to lot
  • Efficiency—detect low-level targets with less sample


How they work

ABfinity™ antibodies are produced by transfecting mammalian cells with high-level expression vectors containing immunogen-specific heavy- and light-chain rabbit antibody cDNA. This highly reproducible process results in consistent lot-to-lot antibody performance.

ABfinity™ oligoclonal antibodies are a mixture of recombinant monoclonal antibodies. This combines the improved signal strength that can come from using polyclonal antibodies, with the highly reproducible results you get from ABfinity™ monoclonal antibodies.

  

ABfinity™ Src recombinant rabbit oligoclonal antibody 
Cytoplasmic localization of Src protein. Immunocytochemistry analysis of HeLa cells probed with ABfinity™ Src recombinant rabbit oligoclonal antibody and visualized using (A) Alexa Fluor® 488 goat anti–rabbit IgG as a secondary antibody (green), (B) DAPI to stain nuclei (blue), and (C) Alexa Fluor® 594 phalloidin to stain actin (red). (D) Composite image of cells showing cytoplasmic localization of Src.



Accurate cholesterol quantitation—MyQubit Amplex® Red Cholesterol Assay

What it is
The MyQubit Amplex® Red Cholesterol Assay for use with the Qubit® 2.0 Fluorometer allows easy and accurate quantitation of cholesterol using a combination of existing Life Technologies™ reagents, common buffers, and the MyQubit Amplex® Red Cholesterol Assay file. The MyQubit Amplex® Red Cholesterol Assay is based on the Amplex® Red Cholesterol Assay Kit, which uses an enzyme-coupled reaction to detect both free cholesterol and cholesteryl esters. This assay is perfect for researchers who only have a few samples, whereas the Amplex® Red Cholesterol Assay Kit is perfect for researchers who have enough samples for a 96-well microplate.



What it offers

  • Sensitivity—accurately detect cholesterol concentrations as low as 200 nM
  • Versatility—perform the reactions in the presence and absence of cholesterol esterase to determine the fraction of cholesterol within a sample that is in the form of cholesteryl esters


How it works

The MyQubit Amplex® Red Cholesterol Assay can detect cholesterol concentrations of 200 nM to 20 mM. The MyQubit Amplex® Red Cholesterol Assay file can be downloaded from the Qubit® 2.0 Fluorometer web page and permanently uploaded to your Qubit® 2.0 Fluorometer. In addition to enabling the MyQubit Amplex® Red Cholesterol Assay, the MyQubit firmware that is preloaded on all new Qubit® 2.0 Fluorometers allows you to create other assays for this fluorometer. Because the instrument is operated by simple commands, creating additional applications can be as straightforward as matching the spectral characteristics of the assay with the right LEDs and emission filters.

 

MyQubit Amplex® Red Cholesterol Assay   The MyQubit Amplex® Red Cholesterol Assay. (A) The plot showing the line corresponding to the curve-fitting algorithm (a modified Hill plot) used to calculate concentration in the MyQubit Amplex® Red Cholesterol Assay. For reference, the positions of the standards and a set of data points from an actual experiment are shown superimposed on the line, demonstrating that the curve-fitting algorithm gives accurate values for quantitation. (B) The assay has the same look and feel as that of the existing Qubit® assays.



Detect reactive oxygen species (ROS) in live cells using flow cytometry—CellROX® Flow Cytometry Assay Kits

What they are
We now offer three new Molecular Probes® kits for oxidative stress detection via flow cytometry. The kits feature CellROX® reagents, probes that become brightly fluorescent when oxidized. Included in each kit is one of the CellROX® reagents formulated for use in flow cytometry, a color-matched dead-cell stain to distinguish dead cells from oxidatively stressed and nonstressed live cells, an antioxidant (N-acetylcysteine [NAC], to serve as a negative control), and an oxidant (tert-butyl hydroperoxide [TBHP], to serve as a positive control).



What they offer

  • Fluorogenic probes—CellROX® reagents become brightly fluorescent when oxidized in cells
  • Multicolor compatibility—minimal overlap with fluorophores excited by other laser lines, allowing easy multiplexing with other reagents
  • Simple—cells can be stained in complete media or other appropriate buffers; no need for serum-free media
  • Fixable—CellROX® Green and CellROX® Deep Red Reagents retain signal following formaldehyde fixation (CellROX® Orange Reagent is not compatible with fixation)


How they work

The three CellROX® reagents all detect ROS in live cells. Each reagent is cell-permeant and is nonfluorescent or very weakly fluorescent in the reduced state. Upon oxidation, the reagents exhibit strong fluorescence and remain localized within the cell. When used together with the SYTOX® Red Dead Cell Stain (or SYTOX® Blue Dead Cell Stain with CellROX® Deep Red Reagent), oxidatively stressed and nonstressed cells are reliably distinguished from dead cells by flow cytometry.

Reactive oxygen species (ROS) detected by flow cytometry 
Reactive oxygen species (ROS) detected by flow cytometry. (A) ROS levels detected by the CellROX® Deep Red Reagent are decreased in TBHP-treated Jurkat cells with pretreatment of cultures using NAC. The cells treated with the oxidant TBHP (red) have increased staining with the CellROX® Deep Red Reagent, compared to the cells pretreated with NAC (blue) and the control cells (green). (B, C) CellROX® Deep Red Reagent can be used in conjunction with SYTOX® Blue Dead Cell Stain to differentiate live stressed cells from dead cells. Jurkat cells were treated with (B) PBS or (C) 200 μM TBHP for 30 minutes before labeling with the CellROX® Deep Red Flow Cytometry Assay Kit. Note that the treated cells (C) have a higher percentage of cells under oxidative stress than the basal level of ROS observed in control cells (B).

CellROX® Flow Cytometry Assay Kits

Product CellROX® detection reagent Ex/Em
maxima (nm)
Dead cell stain Quantity Cat. No.
CellROX® Deep Red Flow Cytometry Assay KitCellROX® Deep Red640/665SYTOX® Blue Dead Cell Stain100 testsC10491
CellROX® Green Flow Cytometry Assay KitCellROX® Green508/527SYTOX® Red Dead Cell Stain100 testsC10492
CellROX® Orange Flow Cytometry Assay KitCellROX® Orange545/565SYTOX® Red Dead Cell Stain100 testsC10493
 



Antibody conjugates for the detection of mouse cell surface markers—new anti–mouse CD conjugates for flow cytometry

What they are
The Molecular Probes® portfolio of over 1,000 highly specific primary antibodies for flow cytometry is expanding to include more Research Use Only (RUO) anti–mouse CD antibodies conjugated to a variety of fluorophores to increase your research options.



What they offer

  • Trusted brand—Molecular Probes® brand
  • Validation—all antibodies are tested in flow cytometry applications
  • Selection—expanded offerings of primary antibody conjugates for flow cytometry


How they work

Life Technologies™ primary antibodies provide direct detection of antigens. All Life Technologies™ antibodies designated “for flow cytometry” have been tested and validated for use in flow cytometry.

 

CD4/L3T4 Rat Anti-Mouse Monoclonal Antibody APC-Cy®7 Conjugate 
Immunofluorescent staining of BALB/c spleen cells for CD4/L3T4. BALB/c spleen cells were stained with CD4/L3T4 Rat Anti-Mouse Monoclonal Antibody APC-Cy®7 Conjugate (0.1 μg/106 cells), gated on small lymphocytes, and analyzed by flow cytometry.

 

NEW APPLICATIONS

A patient-derived stem cell model of hereditary spastic paraplegia with SPAST mutations

Abrahamsen G, Fan Y, Matigian N et al. (2013) Dis Model Mech 6(2):489–502.

In a recent publication, Abrahamsen et al. describe a new cell model of one type of hereditary spastic paraplegia (HSP), a genetically diverse group of diseases that affect long lower nerve fibers, resulting in progressive weakness and spasticity in the legs. The authors took neural progenitor cells derived from the olfactory mucosa of both HSP patients and healthy controls, and compared them to identify cell functions that differ in HSP. Cells were labeled with an HCS CellMask™ stain and analyzed, determining that HSP patient–derived cells are smaller than control cells. MitoTracker® Red CMXRos was used to stain mitochondria, demonstrating abnormally reduced localization in the outer regions of patient-derived cells. CellLight® Peroxisome-GFP, BacMam 2.0 was used to label peroxisomes, showing that HSP affects their intracellular distribution and slows their movement. Patient-derived cells had half the acetylated α-tubulin concentrations of control cells, but treatment with microtubule-binding drugs increased acetylated α-tubulin to normal levels, introducing the possibility of using this cell model to test potential new anti-HSP drug therapies.

PROVEN PERFORMERS

A suite of tools for measuring cell proliferation

Cell proliferation analyses are crucial for cell growth and differentiation studies, and are often used during drug development to evaluate both compound toxicity and inhibition of tumor cell growth.

Proliferation measurements can be based on average DNA content, cellular metabolism, or new DNA synthesis. Various proliferation assays will report live cells counts, total cell numbers, or measure the DNA synthesis in a single cell. The breadth of cell proliferation assays from Life Technologies offers you a range of approaches to give you the right solution for your research.

Proliferation measurement tools:

  • CyQUANT® Assay Kits—microplate assays for DNA content measurement
  • alamarBlue® redox indicator—nontoxic assay for metabolic activity
  • Click-iT® EdU Imaging Kits—DNA synthesis visualized by microscopy or measured with a high-content screening system
  • Click-iT® EdU Microplate Assay—fast and easy DNA synthesis measurement, adaptable to high-throughput systems

Use the selection guides for cell proliferation products to compare product features and review citations from other users.

 

The Click-iT Edu® assay 
The Click-iT® EdU assay is a fast and easy way to measure new DNA synthesis in single cells or cell populations.

 

DEPARTMENTS

On the Web

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Empower your research today using Life Technologies' comprehensive portfolio of products and services to investigate cell signaling pathways and signal transduction—everything from primary antibodies, growth factors, ELISAs, and Luminex® multiplex assays for basic research to assay development, validated biochemical and cell-based assays, and world-class profiling and screening services.

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Mitochondrial and cytoskeletal staining in BPAEC

Mitochondria in bovine pulmonary artery endothelial cells (BPAEC) were labeled with Molecular Probes® MitoTracker® Red CMXRos (yellow). The cells were then fixed and permeabilized. F-actin was stained with Molecular Probes® Alexa Fluor® 488 phalloidin (pink), and nuclei were stained with Molecular Probes® DAPI (blue). Imaging was performed using a Zeiss® LSM 710 Confocal Microscope. Image contributed by Nicholas Dolman, Life Technologies Corporation.

 

Highlight from BioProbes® Journal **

Target key proteins in cell signaling pathways: ABfinity™ recombinant antibodies for cancer research

In the Cell Signaling section of BioProbes 69, you will find the article “Target key protein in cell signaling pathways”, which describes a unique subset of our ABfinity™ recombinant antibodies designed especially for cancer research. These ABfinity™ antibodies, which include phosphorylation site– and acetylation site–specific antibodies, recognize critical proteins in three cell signaling pathways: the p53 signaling pathway, the Akt/mTOR signaling pathway, and the Rb/E2F signaling pathway.

ABfinity™ recombinant antibodies are produced by cloning the heavy and light chains of carefully selected immunoglobulins and then expressing these clones in mammalian cells. Because these recombinant antibodies are produced from renewable and stable clones, they provide peak specificity and sensitivity with every lot. Furthermore, ABfinity™ antibodies are validated for use in specific applications, including immunocytochemistry, ELISA, and western blotting, representing a significant improvement over existing antibodies on the market today.


ABfinity™ recombinant antibodies for cancer research Immunocytochemical analysis using anti–Rb [pT821] ABfinity™ antibodies. U2OS cells were labeled with either (A) anti–Rb [pT821] ABfinity™ recombinant rabbit oligoclonal antibody or (B) anti–Rb [pT821] plus phospho-Rb [pT821] competing peptide. Following the primary antibody incubation, both samples were labeled with Alexa Fluor® 488 goat anti–rabbit IgG secondary antibody (green) as well as with the actin stain Alexa Fluor® 594 phalloidin (red) and DAPI nuclear stain (blue). The composite images show nuclear localization of Rb [pT821]; the lack of antibody signal in cells treated with both the antibody and phosphopeptide demonstrates the antibody’s specificity.

  

** 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!

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