In This Issue

FEATURED NEW PRODUCTS

New PerCP-Cy®5.5 Conjugates for Flow Cytometry Bright Conjugates for 19 Key CD Markers, Stem Cell Markers, and MHC Class II Markers—PerCP-Cy®5.5 Conjugates for Flow Cytometry
   
Total Exosome Isolation Reagent Enrich for Intact Exosomes—Total Exosome Isolation Reagent
   
ABCA4, A2B5, and Rhodopsin New Antibodies to Probe the Retina—ABCA4, A2B5, and Rhodopsin
   
Anti-AGXT, anti-ECH1, anti-ECHD, anti-MFE2, and anti-Catalase Primary Antibodies for Peroxisomal Proteins—Anti-AGXT, Anti-ECH1, Anti-ECHD, Anti-MFE2, and Anti-Catalase
   
New Qdot® Colors Primary Antibody Conjugates for Flow Cytometry—New Qdot® Colors
   
New Antibodies See all of this month's New Antibodies

 

NEW APPLICATIONS

Antibody Master SetLife Technologies and SGC Have Released the First Antibody Master Set for Epigenetics Research

 

PROVEN PERFORMER

CellROX™ Deep Red ReagentVersatile ROS Probe: CellROX® Deep Red Reagent

 UPCOMING MEETINGS

Autumn Immunology Conference (AIC)
November 16–19
Chicago, IL
Booth #12

OTHER PUBLICATIONS

BioProbes® Journal for Cell Biology Applications
BioProbes 68 
 
The Molecular Probes® Handbook
Molecular Probes Handbook
 

 

FEATURED NEW PRODUCTS

Bright Conjugates for 19 Key CD Markers, Stem Cell Markers, and MHC Class II Markers—PerCP-Cy®5.5 Conjugates for Flow Cytometry

What They Are
Antibodies to key CD markers, stem cells markers, and major histocompatibility complex (MHC) class II are now available conjugated to the PerCP-Cy®5.5 fluorophore.



What They Offer

  • Brightness—increased brightness compared to comparable PE-Cy®5.5 conjugates
  • Convenience—packaged in 25 µg or 25 test sizes
  • Additional flexibility—expanded PerCP-Cy®5.5 conjugate offerings to meet your needs


How They Work

PerCP-Cy®5.5 is a tandem conjugate that combines the peridinin chlorophyll protein (PerCP) complexes found in nature with the Cy®5.5 cyanine dye, thus permitting simultaneous multicolor labeling and detection of multiple targets with excitation by a single excitation source—the 488 nm line of the argon-ion laser.

 

Bend.3 mouse endothelial cells stained with CD34 Hamster Anti-Mouse-PerCP-Cy®5.5 Monoclonal Antibody 

 

Bend.3 mouse endothelial cells stained with CD34 Hamster Anti-Mouse-PerCP-Cy®5.5 Monoclonal Antibody.



Enrich for Intact Exosomes—Total Exosome Isolation Reagent

What It Is
Total Exosome Isolation Reagents enable fast and efficient enrichment of intact exosomes from cell culture media or serum samples. The protocol supplied with the reagent is scalable to match your sample size, and the intact exosomes are ready to be used in any type of downstream application.



What It Offers

  • Easy—isolate exosomes using our simple and reliable protocol
  • Fast—avoid time-consuming ultracentrifugation
  • Flexible—can be scaled up or down depending on the sample size


How It Works

By tying up water molecules the reagent forces less-soluble components such as vesicles out of solution, allowing them to be collected using a short, low-speed centrifugation step. Simply add the reagent to the cell culture medium sample, and incubate overnight. Recover the exosomes using standard centrifugation at 10,000 x g for 60 min. You can then resuspend the exosome pellet in buffer for downstream analysis or further purification using affinity methods.

 

Total Exosome Isolation ReagentParticle Analysis of the exosomes recovered from HeLa cell media using Total Exosome Isolation Reagent and the traditional ultracentrifugation protocol. Isolated exosomes were analyzed using a Nanosight LM10 instrument, which uses laser light scattering to track particles’ paths over time to determine their velocity due to Brownian motion. Particle sizes can be calculated from these measurements, independent of density. The above profiles are very finely segmented histograms that indicate the number of particles per mL (in millions) for each size, in 1 nm increment bins from 0 to 1,000 nm. Exosomes recovered with Total Exosome Isolation Reagent have a size distribution very similar to that observed when isolating exosomes using the traditional sucrose gradient ultracentrifugation protocol: all nanoparticles are smaller than 300 nm, most of them about 50 to 150 nm in size.



New Antibodies to Probe the Retina—ABCA4, A2B5, and Rhodopsin

What They Are
Three new mouse monoclonal primary antibodies that recognize retinal proteins offer potentially valuable tools for immunolabeling experiments. The Rim 3F4 IgG clone against ABCA4 is selective for the retina-specific ATP-binding cassette transporter. The 105 IgM clone against A2B5 (derived from chicken embryo retina cells) binds specifically to the cell surface ganglioside GQ1c. The 1D4 IgG1 clone was created using the bovine rhodopsin (~39 kDa) as the immunogen.



What They Offer

  • Anti-ABCA4 and anti-rhodopsin—validated for IHC/ICC and western blotting
  • Anti-A2B5—validated for IHC/ICC and flow cytometry


How They Work

Anti-A2B5 will bind to GQ1c in the retina and the plasma membrane of neurons and islet cells in a wide variety of mammalian species. Antibodies against ABCA4 and rhodopsin can be used in both mammalian and amphibian samples. Used in conjunction with an appropriate secondary detection label, these antibodies can be used to label retinal proteins.

 

immunolabeling of the rhodopsin protein 
Immunohistochemical staining of a mouse retinal section showing specific immunolabeling of the rhodopsin protein in the rod sperules using a monoclonal antibody against mouse rhodopsin. Photo courtesy of Mary Raven, University of California, Santa Barbara, CA.



Primary Antibodies for Peroxisomal Proteins—Anti-AGXT, Anti-ECH1, Anti-ECHD, Anti-MFE2, and Anti-Catalase

What They Offer
Five new mouse monoclonal antibodies provide robust labeling for immunohistochemistry or immunocytochemistry (for both imaging and flow cytometry) and immunoprecipitation. Anti-AGXT, anti-MFE2, and anti-ECH1 have also been validated for in-cell ELISA. These antibodies target antigens typically associated with peroxisomes in a broad range of tissues and cell types.



What They Offer

  • Targeted antigen detection—useful for labeling peroxisomal proteins (unconjugated antibodies require the use of secondary detection labels)
  • Versatile—validated for various applications including IHC, ICC, IP and in-cell ELISA (depends on the antibody)


How They Work

Anti-AGXT, ECH1, ECHD, and MFE2 were derived from human immunogens; anti-catalase was derived from a rat immunogen. All of these primary antibodies against peroxisomal proteins are suitable for use with most mammalian species. In addition to labeling peroxisomal proteins, labeling may also be observed in the mitochondria (depending on the species or disease condition present in the sample).

 

peroxisomal bifunctional enzyme 1 (ECHD) monoclonal antibody 
Immunocytochemistry image of peroxisomal bifunctional enzyme 1 (ECHD) monoclonal antibody. Human HDFn cells were fixed in 4% paraformaldehyde for 20 min and then permeabilized with 0.1% Triton® X-100 for 15 min. The cells were incubated with 1 μg/mL of the antibody overnight at 4°C. Alexa Fluor® 594 goat anti-mouse IgG (H+L) was used as a secondary antibody at a 1:1,000 dilution for 1 hour (red). 10% goat serum was used as the blocking agent for all blocking steps. The cell nuclei were counterstained with DAPI (blue). Target protein locates mainly in peroxisome.



Primary Antibody Conjugates for Flow Cytometry—New Qdot® Colors

What They Are
Qdot® primary conjugates for key human markers are now available in new colors for flow cytometry.



What They Offer

  • Compatibility—combine with existing organic dyes, increase the number of detectable parameters
  • Stability—does not degrade over time like tandem conjugates, affords greater reproducibility
  • Minimal single laser compensation—narrow emission spectra allow for minimal compensation when using a single excitation source


How They Work

Qdot® primary conjugates have extremely bright fluorescence emission that makes them well suited for the detection of low-abundance extracellular proteins. Efficient optical excitation is possible using the 405 nm violet excitation light source. In addition, the narrow, symmetric emission profiles of Qdot® nanocrystal conjugates require substantially lower compensation, enabling better, more efficient multicolor assays using the violet laser.

 

CD45 detection in human lymphocytes 
CD45 detection in human lymphocytes. Histogram overlay plot of gated human lymphocytes, the black line represents cells labeled with CD45RA Mouse Anti-Human mAb (clone MEM-56), Qdot® 800 Conjugate, and the gray line represents unstained cells. Samples were acquired and analyzed using 405 nm excitation and a 780/60 band pass emission filter using the Attune© Acoustic Cytometer Blue/Violet option (Life Technologies, Carlsbad CA).

NEW APPLICATIONS

Life Technologies and SGC Have Released the First Antibody Master Set for Epigenetics Research

First antibody master setLife Technologies recently announced a collaborative partnership with the Structural Genomics Consortium (SGC), a network of more than 200 scientists from both academic and industrial research centers. Their aim is to design and generate the first-ever master set of recombinant epigenetics antibodies for use in disease-related research. Epigenetic regulatory proteins are key targets of drug discovery, and a lack of industry-wide standards for quality antibodies for the study of these proteins has lead to an influx of products on the market that do not perform consistently or with the promised specificity. The recombinant monoclonal antibodies produced from the Life Technologies/SGC partnership address this important deficiency.

PROVEN PERFORMER

Versatile ROS Probe: CellROX® Deep Red Reagent

Oxidative stress results from an imbalance between production of reactive oxygen species (ROS) and the ability of cells to scavenge them. Oxidative stress can be caused by many different pathways, intrinsic and extrinsic, mediated either by mitochondrial respiration or by membrane-bound NADPH oxidases. Detection of ROS by conventional fluorescence microscopy or by high-content imaging is advantageous over other techniques as it gives spatial information and enables multiplexing with other parameters of cell health. CellROX® Deep Red reagent is a fluorogenic probe to measure cellular oxidative stress. The probe is cell permeable and has a peak emission at 665 nm. The dye is non-flourescent in a reduced state and has bright fluorescence when it is oxidized in cells by reactive oxygen species, and the resulting bright fluorescence can be measured by fluorescent imaging (see figure), high-content imaging, fluorescence plate readers, and flow cytometry. In a recent paper, CellROX® Deep Red Reagent was used in cells to show that ROS induction by NADHP oxidases plays an important role in COX-2 expression. (Am J Physiol Lung Cell Mol Physiol 303:L401 (2012)).

 

CellROX™ Deep Red Reagent 
Detection of ROS in bovine pulmonary artery endothelial (BPAE) plated in 96 well plates. BPAE cells were treated with or without 100 µM menadione for 1 hr (left and middle panel). 100 µM of superoxide scavenger, MnTBAP was added to some of the control and menadione-treated wells for the last 30 min of incubation (right panel). The cells were then stained with 5 µM CellROX® Deep Red reagent by adding the probe to the complete media. The cells were then washed with PBS and analyzed on a Thermo Fisher Cellomics ArrayScan® VTI. MnTBAP treatment inhibited oxidative stress caused by menadione, confirming that the signal was due to ROS induced by these compounds.
 

Product Quantity Cat. No.
CellROX™ Deep Red Reagent
10 mLC10422

DEPARTMENTS

On the Web

The World is Your Exosome

 

The World Is Your Exosome

The Life Technologies website now contains a new area devoted to exosome research. Take a look to find products for isolation, characterization, and analysis, links to instructional videos and webinars, and more.

Imaging Corner

ProbesOnline Imaging Corner
Click to enlarge
 

Multiplexing Qdot® Probes, Dyes, and Fluorescent Proteins

U2-OS cells were transduced with CellLight® Plasma Membrane-GFP (green) for 16 h, followed by labeling with Qtracker® 655 Cell Labeling Kit (magenta) for 60 min, and HCS NuclearMask™ Blue (blue). The compiled Z-stack image was acquired on a Zeiss LSM confocal microscope.

From the Bench

Single Molecule Detection Capacity of Qdot® Probes Is Key for Membrane Transporter Studies

Chang JC, Tomlinson ID, Warnement MR et al. (2012) J Neurosci 32(26):8919–8929.

Aberrations in expression or regulation of presynaptic serotonin transporter (SERT) are suspected to contribute to a broad range of neuropsychiatric disorders including anxiety, depression, and autism. Traditional methods to study movement and activation of transporters in cell membranes (i.e., biochemical studies and antibody-based microscopic methods) typically do not provide the resolution needed. By fusing a Qdot® probe to a high-affinity SERT antagonist, Chang and colleagues were able to monitor single SERT proteins on the surface of serotonergic cells. Their results indicated that two SERT pools were present: one that exhibits relatively free diffusion and a second that remains localized to cholesterol-enriched microdomains. The authors postulate that other small-molecule ligands and pharmaceuticals could be conjugated to Qdot® probes using a similar approach, and that this new army of conjugates could be deployed for the investigation of other receptors, ion channels, and transporters at the single-molecule level.

Molecular Probes® Free Webinars

Molecular Probes Webinar Series

 

Flow Cytometry in Microbiological Research

Original broadcast: April 17, 2012
 
In recent years the application of flow cytometry in microbiological research has expanded from detection and quantification of organisms to more complex studies including analysis of host-microbe interactions and detailed spatial and temporal analysis of microbial metabolism in different environments. During this webinar we will discuss how the multi-parametric nature of flow cytometry can be applied to microbiology and the advantages of using this application over traditional microbiological methods.

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