ProbesOnline™ February 2014

In this issue

FEATURED NEW PRODUCTS

  The best are now even better—New Click-iT® Plus EdU proliferation kits for flow cytometry and imaging
     
  Rapid assessment of phagocytosis in whole blood samples—pHrodo™ Green BioParticles® Phagocytosis Kits for flow cytometry
     
  Image single RNA molecules in cells—RNAscope® Fluorescent Multiplex Reagent Kit
     
  Express almost any gene using BacMam technology—ViraPower™ BacMam Expression System
     
  ABfinity™ recombinant antibodies—New antibody for ubiquitin binding protein p62
     
  Isolate intact exosomes—Total Exosome Isolation Reagent

NEW APPLICATIONS

Time-lapse visual monitoring of cell migration and apoptosis during angiogenesis

PROVEN PERFORMERS

  Endogenous background reduction in under an hour—Endogenous Biotin–Blocking Kit

OTHER PUBLICATIONS

BioProbes® Journal of Cell Biology Applications

The Molecular Probes® Handbook

FEATURED NEW PRODUCTS

The best are now even better

New Click-iT® Plus EdU proliferation kits for flow cytometry and imaging

What they are
The next step in cell proliferation assays for flow cytometry and imaging, the Click-iT® Plus EdU proliferation assays provide better performance and an easier workflow than traditional antibody-based BrdU methods. Additionally, the new Click-iT® Plus EdU proliferation assays are compatible with a broader range of commonly used fluorophores, including R-phycoerythrin (R-PE), R-PE tandems, and fluorescent proteins such as GFP.


What they offer

  • Simple and accurate—work more consistently and in less time than traditional methods that use BrdU
  • Multiplex-ready—the method is compatible with simultaneous use of R-PE, R-PE tandems, and fluorescent proteins (e.g., GFP)
  • Robust—enable consistent results; the assay is not dependent on variable antibody lots for detection
  • Efficient—no denaturation steps or harsh treatment required; cell morphology can be preserved

How they work
The new and improved Click-iT® Plus EdU proliferation assays enable faster, more sensitive, reliable detection of cell proliferation, without the need for harsh treatments or antibody-binding steps. Because of the mild reaction conditions, the Click-iT® Plus assays can be used to accurately determine cell proliferation while preserving cell morphology, DNA integrity, antigen-binding sites, and the fluorescence signal from GFP, R-PE, and R-PE tandems. Preservation of DNA integrity allows for DNA staining, including staining with dyes used for cell cycle analysis. 

 

Click-iT® Plus   Cell proliferation detected using the Click-iT® Plus EdU assay vs. a BrdU assay. (A) Erk2-GFP–expressing A375 melanoma cells were treated with the HCl denaturation method required for antibody detection of BrdU incorporation. This treatment results in a loss of GFP signal, and the proliferation signal (BrdU detection) is moderately bright (80 msec exposure). (B) The cells processed using the protocol in the Click-iT® Plus EdU proliferation assay kits retain GFP signal, and the proliferation signal (EdU detection) is very bright (8 msec exposure). High-content analysis was performed using the Thermo Fisher Scientific® Cellomics® ArrayScan® VTI.

Rapid assessment of phagocytosis in whole blood samples

pHrodo™ Green BioParticles® Phagocytosis Kits for flow cytometry

What they are
The pHrodo™ Green E. coli and S. aureus BioParticles® Phagocytosis Kits enable detection of phagocytic activity in whole blood samples and cell lines by flow cytometry without any washing or quenching steps. The kits include all the reagents required for assessing particle ingestion and for red blood cell lysis when starting with whole blood.


What they offer

  • Specific—pH-sensitive fluorogenic dye for detection of phagocytosis
  • Rapid and reproducible—no wash steps or quencher dye required
  • Flexible—multiplex with reagents excited by other laser lines

How they work
pHrodo™ Green conjugates are nonfluorescent outside the cell at neutral pH but fluoresce bright green in acidic environments such as phagosomes. The pHrodo™ Green E. coli and S. aureus BioParticles® conjugates are inactivated, unopsonized Escherichia coli and Staphylococcus aureus, respectively, that function as highly sensitive fluorogenic particles for the detection of phagocytic ingestion. The unique pHrodo™ dye–centered system measures phagocytic activity based on acidification of particles as they are ingested, eliminating the wash and quenching steps that are necessary with nonfluorogenic indicators of bacterial uptake.

 

pHrodo™ Green BioParticles® Phagocytosis Kits

 
pHrodo™ Green E. coli and S. aureus BioParticles® conjugates. Histogram overlays showing the fluorescence separation between experimental samples and negative control samples for (A) pHrodo™ Green E. coli BioParticles® conjugate, and (B) pHrodo™ Green S. aureus BioParticles® conjugate. Samples of whole blood were prepared according to the protocols in the pHrodo™ Green E. coli BioParticles® Phagocytosis Kit for flow cytometry and pHrodo™ Green S. aureus BioParticles® Phagocytosis Kit for flow cytometry and incubated at 37ºC to induce phagocytosis. The samples show an increased fluorescence of the phagocytosed pHrodo™ Green E. coli and S. aureus BioParticles® conjugates (green), in contrast to the negative control samples, which were kept on ice to inhibit phagocytosis (gray). The samples were analyzed on an Attune® Acoustic Focusing Cytometer using a 488 nm laser and 530/30 nm emission filter.  

Image single RNA molecules in cells

RNAscope® Fluorescent Multiplex Reagent Kit

What it is
The RNAscope® Fluorescent Multiplex Reagent Kit enables a novel and proprietary method of in situ hybridization (ISH) that employs signal amplification and background suppression technology to visualize single RNA molecules in samples mounted on slides.


What it offers

  • Simultaneous detection of up to four RNA species
  • Single-molecule detection
  • Ready-to-use reagents
  • Compatibility with fresh tissue and other sample types

How it works
The RNAscope® Multiplex Assay does not require the RNA-free environment used for traditional ISH. It provides exceptional sensitivity, allowing simultaneous single-molecule detection of multiple RNA targets, and is ideal for colocalization studies of any expressed gene set in nearly any tissue type. The RNAscope® Multiplex Assay and Kit were developed by Advanced Cell Diagnostics, Inc., and are distributed by Life Technologies.

Gene-specific custom RNAScope® Probes are required for the RNAScope® Assay but are not included in the RNAScope® Multiplex Reagent Kit. Based on the gene of interest, they can be ordered separately from Life Technologies. Positive and negative probes for common housekeeping genes are also available for ordering.

 

RNAscope® Fluorescent Multiplex Reagent Kit   PGK, PPIB, PolR2A, and HPRT1 expression in human breast cancer tissue. A formalin-fixed, paraffin-embedded human breast cancer section was stained with gene-specific target probes to detect mRNA expression of phosphoglycerate kinase (PGK, green), peptidylprolyl isomerase B (PPIB, pink), RNA polymerase II DNA directed polypeptide A (PolR2A, red), and hypoxanthine phosphoribosyltransferase (HPRT1, yellow), and counterstained with DAPI (blue). The image was obtained using a 40x objective on a Zeiss® Axio® imager and acquired and composited with a Nuance® FX multispectral imaging system.

Express almost any gene using BacMam technology

ViraPower™ BacMam Expression System

What it is
The ViraPower™ BacMam Expression System enables you to express almost any gene of interest in mammalian cells, using BacMam technology. The BacMam system allows expression of genes up to 38 kb, making it possible to clone and express most genes with introns and let the cell do the RNA processing and posttranslational modification. Among all the methods available for transfection, BacMam produces the least cellular toxicity. And because expression is transient and baculoviruses do not replicate in mammalian cells, special containment measures are not required.


What it offers

  • Transiently expresses genes in mammalian cells using BacMam technology
  • Expresses sizes ranging from inserts for RNAi to a large gene with introns (up to 38 kb)
  • Transfects and transduces with almost no cellular toxicity and with maximum safety (nonreplicating in mammalian cells)

How it works
The BacMam pCMV-Dest Vector, included as part of the ViraPower™ BacMam Expression System, combines Gateway® cloning and BacMam gene expression technologies for easy recombination-based cloning and baculovirus-based expression of a target gene in a variety of cell types. In BacMam technology, a modified insect cell virus (baculovirus) is used as a vehicle to efficiently deliver and transiently express genes in mammalian cells with minimum effort and toxicity. This technology allows for control over the level of expression through the increase or decrease of viral particle concentration. When BacMam technology is combined with Gateway® cloning technology, a variety of sizes of target genes can be cloned and expressed. Sizes ranging from an insert for RNAi to a large gene with introns (38 kb) have been cloned and expressed successfully using the BacMam pCMV-Dest Vector. This plasmid accommodates various cloning schemes, including Gateway®, GeneArt® Seamless Cloning and Assembly, and traditional cloning using restriction enzymes.

 

BacMam technology   BacMam-mediated gene delivery. BacMam particles are taken up by endocytosis and their contents released for transcription and expression (following migration to the nucleus). Gene expression begins within 4 to 6 hours of transduction and nears its maximum level within 24 hours of transduction.

ABfinity™ recombinant antibodies

New antibody for ubiquitin binding protein p62

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.

p62 is a scaffold protein that plays an important role in autophagy by acting as a link in recognizing ubiquitinated targets and presenting them to autophagic machinery. The N-terminus of p62 aids in polymerization, while the C-terminus aids in binding to ubiquitinated proteins and to autophagosomal proteins, such as LC3. p62 is ubiquitously expressed in adult tissues, where it colocalizes with ubiquitinated proteins in protein bodies found in the cytosol and nucleus or within autophagosomes and lysosomal structures.


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 chain 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 can get from ABfinity™ monoclonal antibodies.

Both of the anti-p62 ABfinity™ antibody offerings are validated for use in western blotting, indirect ELISA, and immunocytochemistry.

 

 
Immunocytochemistry analysis of p62 expression in HeLa cells. Cells were treated with chloroquinone and probed with p62 ABfinity™ Recombinant Rabbit Monoclonal Antibody (clone 11HC14LC25). (A) Alexa Fluor® 488 goat anti–rabbit IgG was used as secondary antibody (green). (B) DAPI was used to stain the nucleus (blue). (C) Composite image of cells showing cytoplasmic localization of p62.

Isolate intact exosomes

Total Exosome Isolation Reagents

What it is
Now you can easily enrich for intact exosomes from a variety of starting samples, using a method that is much easier and less tedious than ultracentrifugation. We provide Total Exosome Isolation products optimized for enriching exosomes from cell culture media, serum, plasma, urine, or other body fluids.


What it offers

  • Typically 15–20 minutes of hands-on time
  • Simple and reliable protocols that can be scaled to a range of sample sizes
  • Compatible with any type of downstream application

How it works
The flexible and scalable Total Exosome Isolation Reagents allow for fast and efficient exosome enrichment. After an overnight incubation at 2–8°C, exosomes can be recovered using a standard benchtop centrifuge to spin them at 10,000 x g for 60 minutes. Simply resuspend the pellet, and the exosomes are ready for downstream analysis. 

 

Exosomes    
     
Analysis of exosomes recovered from HeLa cell media. (A) Exosomes recovered with Total Exosome Isolation Reagent (from cell culture media) have a size distribution comparable to (B) exosomes isolated following a traditional sucrose gradient ultracentrifugation protocol. Profiles analyzed on a NanoSight® LM10 instrument show all particles to be smaller than 300 nm; most are about 50–150 nm in size.    

NEW APPLICATIONS

Time-lapse visual monitoring of cell migration and apoptosis during angiogenesis

During the formation of blood vessels, endothelial cells undergo complicated, coordinated movements to form the tubular vessel structures, while some neighboring cells that are not needed in the process undergo apoptosis and are eliminated. We have combined Molecular Probes® reagents, Gibco® cell culture products, and the EVOS® FL Auto Imaging System to visualize cells as they form pseudo-tubes or undergo apoptosis in a model of angiogenesis.

The EVOS® Onstage Incubator is an environmental chamber that enables precise control of temperature, humidity, and three gases for time-lapse imaging of live cells under either physiological or nonphysiological conditions. It was designed specifically for the EVOS® FL Auto Imaging System, so control of all environmental parameters is seamlessly integrated with the EVOS® FL Auto user interface. Equipped with the EVOS® Onstage Incubator, the EVOS® FL Auto Imaging System is exquisitely suited to investigating processes of developmental morphogenesis that occur over periods of minutes, hours, or even days.

In the video that follows, CellTracker™ Red CMTPX, a red-fluorescent cell tracer, was used to label cultured endothelial cells and follow their migration in an in vitro model of angiogenesis over a period of 40 hours. The cells were also incubated with CellEvent® Caspase-3/7 Green ReadyProbes® Reagent, an indicator that is internalized by cells and fluoresces green when cleaved by activated caspase-3 or caspase-7, early markers of apoptosis.

Tracking pseudo-tube formation and apoptosis during angiogenesis. Human umbilical vein endothelial cells (HUVEC) were plated on a thin layer of Geltrex® basement membrane matrix at 40,000 cells/cm2. Adhered cells were labeled with 1 µM CellTracker™ Red CMTPX in unsupplemented Gibco® Medium 200 for 30 min. The medium was then replaced with fresh Medium 200 containing Large Vessel Endothelial Supplement (LVES) and CellEvent® Caspase-3/7 Green ReadyProbes® Reagent. Samples were imaged on the EVOS® FL Auto Imaging System equipped with the EVOS® Onstage Incubator. Time-lapse imaging every hour for 40 hours with a 4x objective shows cells migrating and differentiating (red) to form blood vessel–like structures. Undifferentiated and unneeded cells undergoing apoptosis appear green.

PROVEN PERFORMERS

Endogenous background reduction in under an hour

Endogenous Biotin–Blocking Kit

The method and reagents provided in the Endogenous Biotin–Blocking Kit are for a pretreatment that reduces or eliminates background signals when biotin-avidin or biotin-streptavidin detection systems are used to identify cellular targets.

The Endogenous Biotin–Blocking Kit offers:

  • Improved results—increased signal-to-noise ratio facilitates cell target identification
  • Ease of use—pipette-free dropper bottle offers speed and convenience
  • Flexibility—the kit can be used to help improve results from conventional immunohistochemistry, immunocytochemistry, RNA FISH, and ELISA procedures

Biotin occurs naturally, functioning as an enzyme cofactor in the cytosol and mitochondria of a wide variety of cell types. The Endogenous Biotin–Blocking Kit minimizes interference from the endogenous biotin found in enzymes of mammalian cells and tissues, through a two-step blocking strategy. First, an excess of unlabeled streptavidin is added to the specimen to bind endogenous biotin-rich enzymes. The streptavidin is subsequently blocked with an excess of unlabeled biotin, effectively rendering the cell or tissue sample free of available biotin-binding sites.


Endogenous Biotin–Blocking Kit
  Reducing endogenous background with the Endogenous Biotin–Blocking Kit. A549 cells were fixed and permeabilized, treated (A) or not (B) with the Endogenous Biotin–Blocking Kit, labeled with a primary antibody against mouse golgin-97 followed by DSB-X biotin–conjugated goat anti–mouse IgG, then stained with Alexa Fluor® 488 streptavidin (green), Alexa Fluor® 647 phalloidin (magenta), and NucBlue® Fixed Cell ReadyProbes® Reagent (blue). Note the green fluorescence produced by the labeled streptavidin is punctate and limited to the target Golgi apparatus in the treated cell (A), but there is considerable diffuse, nonspecific green fluorescence in the untreated cell (B), in addition to the punctate staining. The image was acquired at 60x magnification with a Nikon® E800 upright microscope.

DEPARTMENTS

On the web

Stem cell breakthrough—Corneal cells from hair follicles

After 10 years of research, the Stem Cell Team at the Saint-Louis Hospital (Paris, France), directed by Dr. Daniel Aberdam, has succeed in creating corneal cells from hair cells. The team collected a hair, cultured the hair cells, and managed to reprogram them to form corneal cells.

The EVOS® XL Imaging System is a fully integrated digital, transmitted-light, inverted-imaging system that combines advanced ergonomic design, an on-board microprocessor, LED illumination, sensitive color camera, and a highly streamlined user interface, to deliver superior flexibility and ease of use. The EVOS® XL Imaging System is ideal for both demanding and routine cell culture and colorimetric sample imaging applications.

Gauge fluorophore facts at a glance—Fluorophore dashboards

Looking for the best fluorophore for your application? Molecular Probes® fluorophore dashboards provide all the information you need to make the right selection. Fundamental properties like brightness, photostability, excitation and emission maxima, and optimal laser lines and filter sets are grouped in a standardized display for each fluorophore, to make its performance easy to visualize and compare to other fluorophores. Find the stain index for a flow cytometry label, see photostability with antifade treatment for an imaging probe, or instantly find out if a fluorophore is well-matched to your instrument’s laser light source.    

Each fluorophore summary page features a dashboard and is conveniently linked to reactive dye forms, labeling kits, and conjugates to help you find the correct fluorescent product for your application.

Imaging corner

Mitochondria, actin, and nuclei highlighted by fluorescent staining of HeLa cells

Enlarge Image  
HeLa cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit, then incubated with Anti–ATP Synthase Subunit IF1 and fluorescein-conjugated goat anti–mouse IgG secondary antibody. Cells were counterstained with NucBlue® Live ReadyProbes® Reagent and ActinRed™ 555 ReadyProbes® Reagent, then mounted using ProLong® Gold Antifade Reagent and imaged with the EVOS® FL Auto Imaging System with a 40x coverslip-corrected objective.

Highlight from BioProbes® Journal

Expand your multicolor capacity with the Pacific Green™ dye: Pacific Green™ dye and conjugates for flow cytometry

In the Flow Cytometry section of BioProbes® 69, you will find the article “Expand your multicolor capacity with the Pacific Green™ dye”, which describes the violet light–excitable Pacific Green™ dye and its conjugates. Pacific Green™ dye joins Pacific Orange™ and Pacific Blue™ dyes, which are established violet laser–excitable fluorophores for flow cytometry. Pacific Blue™, Pacific Green™, and Pacific Orange™ dye conjugates can be simultaneously excited at 405 nm for emission at 455 nm, 500 nm, and 551 nm, respectively, enabling three-color analysis with a single laser line. When used for three-color immunophenotyping, these Pacific dyes require only minimal compensation and exhibit very little 488 nm cross-excitation.

By including violet light–excitable fluorophores in your flow cytometry experiments, you can free up the 488 nm and 635 nm laser lines for detection of more specialized markers, for which blue light– and red light–excitable fluorescent conjugates are more likely to be commercially available. The Pacific Green™ dye is available in several formats to fit most flow cytometry applications. These include:

  • Pacific Green™ primary and secondary antibody conjugates
  • Pacific Green™ streptavidin
  • Zenon® Pacific Green™ Mouse IgG1 Labeling Kit, for creating direct primary antibody conjugates
  • Amine-reactive Pacific Green™ succinimidyl ester, for covalently labeling accessible primary amines

Read the full article
Subscribe to BioProbes® Journal

 

 
   
Bivariate plot displays for multicolor immunophenotyping by flow cytometry. Normal human blood was labeled with four different fluorophore-conjugated primary antibodies. Lymphocyte gating with the Pacific Orange™ conjugate of anti–human CD45 antibody (A) was used as the parent for the subsequent bivariate plots. Fluorescence of the Pacific Green™ conjugate of anti–human CD3 antibody was plotted against the fluorescence of the Pacific Blue™ conjugate of anti–human CD8 antibody (B) and the Alexa Fluor® 488 conjugate of anti–human CD56 antibody (C). Data acquisition was performed on the Attune® Acoustic Focusing Cytometer with the violet (405 nm) and blue (488 nm) laser options and the standard event collection rate of 100 μL/min; data were analyzed using Attune® Software.  

† 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 the current BioProbes® Journal take shape!