In This Issue

FEATURED NEW PRODUCTS

ABfinity™ Recombinant Antibodies New Tools for Vascular Studies—ABfinity™ Recombinant Antibodies for P-Selectin
   
Markers for Flow Cytometry New Murine CD Markers for Flow Cytometry—Rat Anti-Mouse mAb Conjugates
   
Multiplex Rat Cytokine Markers Multiplex Rat Cytokine Markers in One Well—Rat Cytokine Magnetic 10-Plex Panel
   
New Antibodies See All of This Month's New Antibodies


NEW APPLICATIONS

CyQUANT® Direct AssayCyQUANT® Direct Assay for High-Throughput Screening of Cell Proliferation and Cytotoxicity

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FEATURED NEW PRODUCTS

New Tools for Vascular Studies―ABfinity™ Recombinant Antibodies for P-Selectin

what they are
P-selectin is a 140 kDa type 1 transmembrane glycoprotein stored in the Weibel-Palade bodies of endothelial cells, as well as in the α-granules of platelets. This protein can rapidly be brought to the cell surface after exposure to thrombin, histamine, complement 5a, Ca21 ionophores, or adenosine diphosphate. Researchers have found that P-selectin, which is encoded by the SELP gene, contributes to the adverse vascular processes that promote cognitive impairment in individuals with cardiovascular disease. Our broad antibody portfolio now includes ABfinity™ recombinant monoclonal and oligoclonal antibodies for P-selectin.



what they offer

  • Consistent lot-to-lot results
  • Minimize the need to revalidate working antibody dilutions each time you order


how they work

ABfinity™ antibodies are manufactured by transfecting mammalian cells with high-level expression vectors containing immunogen-specific antibody heavy- and light-chain cDNA. This production process offers consistent lot-to-lot antibody performance. ABfinity™ oligoclonal antibodies are a mixture of recombinant monoclonal antibodies. This combines the improved signal strength of a polyclonal antibody with the highly reproducible results you get from ABfinity™ monoclonal antibodies. 

 

U2OS cells stained with P-Selectin ABfinity™ Recombinant Rabbit Monoclonal Antibody 
Immunocytochemistry analysis using P-Selectin ABfinity™ Recombinant Rabbit Monoclonal Antibody. U2OS cells were stained with P-Selectin ABfinity™ Recombinant Rabbit Monoclonal Antibody. (A) Cells were labeled with Alexa Fluor® 488 goat anti-rabbit secondary antibody (green). (B) Nuclei were stained with DAPI (blue). (C) Actin was stained with Alexa® Fluor 594 phalloidin (red). (D) The composite image shows the membrane localization of P-selectin.



New Murine CD Markers for Flow Cytometry―Rat Anti-Mouse mAb Conjugates

what they are
Rat anti-mouse CD90 (clone G7) and CD90.2 (clone 30-H12) monoclonal antibodies are now available as FITC, phycoerythrin (PE), and allophycocyanin (APC) conjugates.



what they offer

  • Flexibility—3 colors available for use with a blue or red laser
  • Convenience—packaged in 25 µg size
  • Affordability—average price is 50% less than other antibodies


how they work

Part of the immunoglobulin superfamily, CD90/Thy-1 is a GPI-anchored cell surface receptor expressed on thymocytes, peripheral T lymphocytes, some intraepithelial T lymphocytes, and neurons of all mouse strains, whereas the CD90.2/Thy-1.2 alloantigen is expressed on all thymocytes, peripheral T lymphocytes, and some intraepithelial T cells of most mouse strains. The CD90/Thy-1 clone G7 antibody recognizes the Thy-1.1 and Thy-1.2 alloantigens (Thy-1 epitope region A). The CD90.2/Thy-1.2 clone 30-H12 antibody recognizes the mouse CD90.2/Thy-1.2 alloantigen.

 

stained BALB/c splenocytes 

 

BALB/c splenocytes were double-stained with rat anti-mouse CD90.2-FITC and rat anti-mouse CD19-RPE.



Multiplex Rat Cytokine Markers in One Well―Rat Cytokine Magnetic 10-Plex Panel

what it is
The Rat Cytokine Magnetic 10-Plex Panel for the Luminex® platform is designed to simultaneously quantify 10 rat cytokines in serum, plasma, and tissue culture supernatant samples. The magnetic beads facilitate automation and easier wash steps. The panel is suitable for use with the Luminex® 200™, FLEXMAP 3D®, and MAGPIX® systems.



what it offers

  • Superior performance—accurate, reproducible, and sensitive quantitation of multiple proteins
  • More from your precious samples―measure multiple proteins in the same sample
  • Fast and easy protocols—perform your assays and analyze your data typically in less than one day


how it work
s
The assay has a 3.5-hour incubation time and is similar to an ELISA workflow. Magnetic beads covalently bound to different antibodies can be mixed in the same assay in a 96-well microplate format. At the completion of the sandwich immunoassay, beads can be read using a Luminex® instrument and xPONENT® software to distinguish bead color (analyte) and assay signal strength (PE fluorescence intensity).

 

Standard curves for protein targets 


Typical standard curves of protein targets depicted in two groupings for clarity.

Product Quantity Cat. No.
Rat Cytokine Magnetic 10-Plex Panel100 testsLRC0002M

 

NEW APPLICATIONS

CyQUANT® Direct Assay for High-Throughput Screening of Cell Proliferation and Cytotoxicity

The CyQUANT® Direct Cell Proliferation Assay is based on a cell-permeant DNA-binding dye that is used in combination with a background suppression reagent. DNA content is highly regulated, and cell number estimates obtained with the CyQUANT® Direct assay are very accurate. As a result, the CyQUANT® Direct assay is commonly used as a fluorescence-based readout for cell proliferation and cytotoxicity. The addition-only, no-wash assay format renders the CyQUANT® Direct assay suitable for high-throughput screening (HTS) applications.

A new protocol provides guidance for using the CyQUANT® Direct assay with mammalian cells in HTS applications. Because the CyQUANT® Direct assay readout involves highly sensitive fluorescence measurement of cellular DNA content, the protocol provides recommendations for proper plate setup and handling that are essential for optimal assay performance.

 

HeLa cells 
Accurate cell number estimates obtained with the CyQUANT® Direct assay. HeLa cells were serially diluted and plated in 384-well microplate format. Data are the mean ± standard deviation of cell plating density (n = 10 replicate wells).

 

CyQUANT® Direct Even distribution of the cells in the wells is important for optimal CyQUANT® Direct assay performance. (Left) Cells stained with the CyQUANT® Direct Cell Proliferation Assay that were not set up properly for high-throughput screening and as a result grew unevenly during a cell proliferation time course. (Right) Cells that were set up correctly according to protocol recommendations, resulting in even distribution across the well.

 

DEPARTMENTS

On the Web

 

Watch the MAGPIX® System in Action

An affordable, compact fluorescence detection system based on proven Luminex® technology, the MAGPIX® multiplexing system provides an all-in-one solution for quantitative protein analysis.

Imaging Corner

Imaging oxidative stress with CellROX® Green Reagent Enlarge Image  

Imaging Oxidative Stress with CellROX® Green Reagent

U2OS cells were treated with 100 µM menadione for 1 hr. A staining solution containing 5 µM of CellROX® Green Reagent, 300 nM of  MitoTracker® Deep Red FM, and 2 drops/mL of NucBlue™ Live Cell Stain was applied for 30 min at 37°C. CellMask™ Orange Plasma Membrane Stain (5 µg/mL) was added directly to the cells for the last 5 min of incubation. Cells were washed and imaged with Live Cell Imaging Solution. Image contributed by Kary Oakleaf and Bhaskar Mandavilli, Life Technologies.

 

From the Bench

Rapid Click Chemistry Labeling Compatible With Live Cells

Uttamapinant C, Tangpeerachaikul A, Grecian S et al. (2012) Angew Chem Int Ed 51:5852–5856.

The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is commonly used for biomolecule conjugation but poses toxicity problems when used to label molecules in living cells because of the reactive oxygen species generated by the relatively high levels of CuI needed for reaction efficiency. Uttamapinant and colleagues have developed a new azide reaction partner—a picolyl azide, containing an internal CuI ligand—that maintains an increased CuI concentration in the region of the reaction site only. After only 20 minutes of incubation time in the presence of a noncytotoxic concentration of CuSO4 (50 µM), this picolyl azide delivered improved labeling of the target biomolecules in live cells (up to 25-fold better than the alkyl azide), and was even effective for labeling delicate neurons. In further experiments to investigate its utility for visualizing RNA and proteins metabolically labeled with 5-ethynyl uridine and L-homopropargylglycine, respectively, the picolyl azide delivered ~2-fold improvement in signal intensity compared to the alkyl azide form.

Molecular Probes® Free Webinars

Molecular Probes Webinar Series

 

A Practical Approach to Antibody Labeling

The growing number of available fluorophores makes labeling your own antibodies a tempting proposition. But with so many antibody labeling solutions available, selecting the best option can be a daunting task. This webinar will provide you with:

  • An overview of our antibody labeling kits
  • Guidance on which methods are ideal for specific applications and experiments
  • Tips and tricks to optimize your labeling protocol

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