In This Issue

FEATURED NEW PRODUCTS

EVOS® Cell Imaging Systems Even More to Love—Meet the Whole Family of EVOS® Cell Imaging Systems
   
CellEvent® ReadyProbes™ Reagent Apoptosis Detection Has Never Been Easier—CellEvent® Caspase-3/7 Green ReadyProbes™ Reagent
   
SiteClick™ Antibody Labeling Kits Site-Specific Antibody Labeling in 3 Easy Steps—SiteClick™ Antibody Labeling Kits
   
MyQubit miRNA Assay Accurate microRNA Quantitation —MyQubit miRNA Assay
   
ABfinity™ Antibodies for CD44 ABfinity™ Recombinant Antibodies—New Antibodies for CD44
   
New Antibodies See All of This Month's New Antibodies

 

NEW APPLICATIONS

MacroautophagyLive Cell Analysis of Clearance of Polyubiquitinated Cargo by Macroautophagy

 

PROVEN PERFORMERS

Quant-iT™ and Qubit® Broad Range RNA Assays Quant-iT™ and Qubit® Broad Range RNA Assays Deliver Accurate mRNA Quantitation

 UPCOMING MEETINGS

American Association of Immunology (AAI)
May 3–7   |   Honolulu, HI
Booth #1729

International Society for Advancement of Cytometry (ISAC/CYTO)
May 19–23  |   San Diego, CA
Booth #624

American Society for Microbiology 112th General Meeting (ASM)
May 18–21   |   Denver, CO
Booth #736

International Society for Stem Cell Research (ISSCR)
June 12–15   |   Boston, MA
Booth #630

OTHER PUBLICATIONS

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

FEATURED NEW PRODUCTS

Even More to Love—Meet the Whole Family of EVOS® Cell Imaging Systems

What They Are
Minimize the complexities of microscopy without compromising performance. No matter your application or budget, there’s an EVOS® cell imaging instrument to bring simplicity right to your bench.



What They Offer

  • Convenient—place right on the bench, not in the darkroom
  • Economical—LED light sources (>50,000-hour life) reduce cost of replacing bulbs
  • Simple—intuitive user interface allows novices or experts to generate images in a snap


How They Work

Combining the EVOS® cell imaging systems with the FLoid® Cell Imaging Station, Life Technologies provides cell imaging systems that minimize the complexities of capturing and processing cell images, allowing researchers to focus on analyzing data rather than worrying about microscope operation. From initial cell culture checks (viability and morphology) to more complex fluorescent analysis (time-lapse and image stitching), EVOS® cell imaging systems provide simple, intuitive, and fast imaging solutions without compromising image quality.

 

EVOS® Cell Imaging Systems
 
U2OS cells stained with the reagents in the LIVE/DEAD® Cell Imaging Kit. Live cells exhibit green fluorescence, and dead cells (with compromised membranes) exhibit red fluorescence. After staining, cells were treated with 1 µM staurosporine, which initiates cell death. Fluorescence images in the FITC and Texas Red® channels were acquired every 5 minutes over 14 hours on the EVOS® Auto Cell Imaging System, using a 20x objective.



Apoptosis Detection Has Never Been Easier—CellEvent® Caspase-3/7 Green ReadyProbes™ Reagent

What It Is
CellEvent® Caspase-3/7 Green ReadyProbes™ Reagent is a fluorogenic, no-wash indicator for activated caspase-3/7, an early indicator of apoptosis. Like other ReadyProbes™ reagents, CellEvent® Caspase-3/7 Green reagent is formulated in a room temperature–stable solution and comes in a convenient dropper bottle. Just tip and drip two drops per milliliter to measure apoptosis.



What It Offers

  • Optimized caspase-3/7 substrate for apoptosis analysis
  • Simple and fast no-wash protocol helps preserve delicate apoptotic cells
  • Ready-to-use liquid formulation in convenient dropper bottle—no need to dilute, weigh, or pipet


How It Work
s
The CellEvent® Caspase-3/7 Green reagent is a 4–amino acid peptide (DEVD), conjugated to a nucleic acid–binding dye that is nonfluorescent when not bound to DNA. Apoptosis-related caspase induction specifically cleaves the DNA-binding moiety, resulting in fluorescence. The assay requires no wash, reducing the risk of detaching fragile apoptotic cells. The CellEvent® Caspase-3/7 Green ReadyProbes™ assay is therefore highly accurate and robust. Importantly, the signal survives fixation and permeabilization, allowing endpoint assays and immunocytochemistry-based methods.

 

CellEvent™ Caspase 3/7 Green ReadyProbes™ reagent

 
CellEvent® Caspase-3/7 Green ReadyProbes™ Reagent.
HeLa cells were loaded with CellEvent® Caspase-3/7 Green ReadyProbes™ reagent, then treated with 0.5 μM staurosporine or vehicle control for 4 hours.
(A) Untreated cells have minimal fluorescence. (B) Treated cells have a significant increase in fluorescence (green), indicating apoptosis.



Site-Specific Antibody Labeling in 3 Easy Steps—SiteClick™ Antibody Labeling Kits

What They Are
The SiteClick™ Antibody Labeling Kits allow you to conjugate your IgG antibodies to Qdot® particles (525, 565, 585, or 605 nm emission) or DIBO‑modified R-phycoerythrin (R-PE).



What They Offer

  • Simplicity—in 3 simple steps, expertly attach Qdot® particles or R-PE to your antibody
  • Quality conjugates—enzymatic labeling system keeps antibodies and antigen-recognition site intact


How They Work

In the first step of SiteClick™ conjugation, the Fc region of the antibody is modified by β-galactosidase.
The azide-containing sugar UDP-GalNAz is then added to the modified carbohydrate domain of the antibody via the β-1,4-galactosyltransferase (GalT)-catalyzed reaction targeting the specific N-linked oligosaccharide GlcNAc residues. This specific targeting maintains the integrity of the antigen-binding site on the antibody. Finally, the antibody (now containing an azide moiety) is conjugated to the DIBO-modified label (Qdot® particle or R-PE) in a copper-less click reaction with a simple overnight incubation.

 

SiteClick™ conjugation reaction
Click to enlarge
 
SiteClick™ conjugation reaction.
 



Accurate microRNA Quantitation—MyQubit miRNA Assay

What It Is
The MyQubit miRNA Assay for use with the Qubit® 2.0 Fluorometer allows easy and accurate quantification of miRNA using a combination of existing Life Technologies reagents, common buffers, and the MyQubit miRNA Assay file. The MyQubit miRNA Assay is based on the Quant-iT™ OliGreen® ssDNA Reagent, which exhibits a large increase in fluorescence upon binding to nucleic acids.



What It Offers

  • Sensitivity—accurately detect as little as 0.5 ng
  • Simplicity—add your sample (any volume between 1 μL and 20 μL), then read the concentration on the Qubit® 2.0 Fluorometer


How It Works

The MyQubit miRNA Assay has a core dynamic range of 5–500 ng/mL miRNA (see figure). The MyQubit miRNA Assay file can be downloaded and permanently uploaded to your Qubit® 2.0 Fluorometer. In addition to the MyQubit miRNA Assay, the MyQubit firmware preloaded on all new Qubit® 2.0 instruments allows you to create other assays for your Qubit® 2.0 Fluorometer. Since the instrument is operated by simple commands, creating additional applications can be as straightforward as matching spectral compatibility with the LEDs and emission filters.

 

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



ABfinity™ Recombinant Antibodies—New Antibodies for CD44

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. Over the past year we have launched several new ABfinity™ recombinant antibodies.

CD44 is a glycoprotein expressed on the cell membrane. CD44 is a receptor for HA with good affinity to osteopontin, collagens, and MMP. CD44 plays an essential role in many diverse functions such as cell proliferation, differentiation, migration, tumor growth, angiogenesis, presentation of cytokines or growth factors to their corresponding receptors, hematopoiesis, lymphocyte activation, recirculation, and homing. CD44 exhibits high heterogeneity because of its abundant alternative splicing, varied posttranslational modifications, and numerous natural variants. One such variation in position 46 is responsible for the Indian blood group system.



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 manufactured by transfecting mammalian cells with high-level expression vectors containing immunogen-specific heavy- and light-chain antibody 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 that can come from using a polyclonal, with the highly reproducible results you get from ABfinity™ monoclonal antibodies.

 

ABfinity™ Recombinant Antibodies 
Immunocytochemistry analysis of HeLa cells stained with CD44 ABfinity™ Recombinant Rabbit Oligoclonal Antibody. (A) Alexa Fluor® 488 goat anti–rabbit IgG was used as the secondary antibody (green), (B) DAPI was used to stain the nucleus (blue), and (C) Alexa Fluor® 594 phalloidin was used to stain actin (red). (D) Composite image of the cells, showing cytoplasmic localization of lipocalin-1.

 

NEW APPLICATIONS

Live Cell Analysis of Clearance of Polyubiquitinated Cargo by Macroautophagy

p62, also known as SQSTM1, contains a ubiquitin-binding domain (UBD) and an LC3-interacting region (LIR). The concerted action of these two domains brings polyubiquitinated cargo (via the UBD) into the autophagy pathway (interacting with the key autophagy protein LC3 via the LIR), leading to degradation of these structures in the lysosome. Premo™ Autophagy Sensor RFP-p62 and GFP-p62 are fluorescent protein–based biosensors that report the formation of p62-positive aggregates within the cytoplasm. Used in conjunction with LysoTracker® Deep Red or Premo™ Autophagy Sensor LC3B-GFP (or -RFP), Premo™ Autophagy Sensor RFP-p62 and GFP-p62 give context-rich information on the recruitment of p62 into autophagosomes followed by the lysosome. And because Premo™ sensors are delivered using highly efficient BacMam 2.0 technology, researchers can study these processes in a broad range of cell types, including primary human cells, neurons, and stem cells.

 

Time-lapse imaging of GFP-p62–positive aggregates in A549 cells
Time-lapse imaging of GFP-p62–positive aggregates in A549 cells during nutrient deprivation and chloroquine-mediated autophagy block. A549 cells were transduced with Premo™ Autophagy Sensor GFP-p62 (40 particles/cell) and cultured for 48 hours. Cells were incubated in Earle's Balanced Salt Solution containing 120 µM chloroquine, and images were acquired every 2 minutes. A selection of shows the accumulation of p62-positive aggregates over time.

PROVEN PERFORMERS

Quant-iT™ and Qubit® Broad Range RNA Assays Deliver Accurate mRNA Quantitation

Accurate quantitation of nucleic acid samples is critical for high-value downstream applications such as qPCR and RNA-Seq. The Quant-iT™ and Qubit® broad range (BR) assay kits make it simple to quantitate samples following RNA purification (e.g., using the Dynabeads® mRNA DIRECT™ Micro Kit). Unlike UV-based detection methods, the Quant-iT™ and Qubit® BR assays are highly selective for RNA over double-stranded DNA (dsDNA), do not detect RNA that has degraded into free nucleotides, and are accurate for initial sample concentrations of 1–1,000 ng/μL, providing an assay range of 20–1,000 ng. The assays are performed at room temperature, and the signal is stable for 3 hours. The Qubit® RNA BR assay is designed for use with the Qubit® Fluorometer and is ideal for <20 assays/day. The Quant-iT™ RNA BR assay is suited to higher throughput (20–2,000 samples) and compatible with standard filters on fluorescence microplate readers.

 

Performance of the Qubit® BR RNA Assay 


Performance of the Qubit® RNA BR Assay. The Qubit® RNA BR Assay has a linear detection range of 20–1,000 ng and is selective for RNA, even in the presence of an equal mass of DNA.

Product Quantity Cat. No.
Qubit® RNA BR Assay Kit1 kit, 100 assaysQ10210 
1 kit, 500 assaysQ10211
Quant-iT™ RNA Assay Kit, Broad Range1 kitQ10213
Qubit® 2.0 Fluorometer1 instrumentQ32866
Qubit® Assay Tubes1 set, 500 tubesQ32856
Dynabeads® mRNA DIRECT™ Micro Kit1 kit61021

 

DEPARTMENTS

On the Web

CellEvent® Caspase-3/7 Green Detection Reagent 

Researchers Are Bringing Cell Death to Light

Since its release, CellEvent® Caspase-3/7 Green Detection Reagent has been used by researchers like you as a simple, effective method for monitoring apoptosis in live  and fixed cell samples. See the rapidly growing list of citations to see how researchers have used CellEvent® Caspase-3/7 Green Detection Reagent in their experimental systems.

Imaging Corner

EVOS® Auto Imaging System
Click to enlarge
 

Time Course of Cell Death Visualized Using the LIVE/DEAD® Cell Imaging Kit

The LIVE/DEAD® Cell Imaging Kit is based on a cell-permeant dye (calcein AM) that stains live, viable cells bright green, and a cell-impermeant red marker that only stains dead and dying cells, characterized by compromised cell membranes. Labeled U2OS cells were treated with 1 μM staurosporine, and fluorescence images were acquired in the FITC and Texas Red® channels on the EVOS® Auto Cell Imaging System using a 20x objective.

 

Highlight From BioProbes® Journal

Autophagy Made Visible

In the Cell Signaling section of BioProbes 69 you will find “Autophagy made visible,” an article describing a suite of advanced fluorescent probes specifically designed for the study of the two major catabolic processes in mammalian cells: the ubiquitin-proteasome system (UPS) and the autophagy pathway. The Click-iT® HPG labeling method provides a click chemistry–based approach for measuring protein lifetimes and for imaging protein aggregates that accumulate when either proteasome or autophagic activity is blocked. The state-of-the-art Premo™ Autophagy Sensors are fluorescent protein chimeras of p62 or LC3B for visualizing autophagosome function in real time. And don’t forget to check out our newest lysosome marker—LysoTracker® Deep Red dye—with long-wavelength emission that is ideal for four-color imaging with GFP, RFP, and a blue-fluorescent nuclear counterstain.


Autophagy Made Visible 
Protein accumulation as a result of inhibiting normal protein clearance by the autophagy pathway.
Blockage of the autophagy pathway with chloroquine causes protein accumulation (green), revealed by detection of HPG-containing proteins with the Click-iT® HPG Alexa Fluor® 488 Protein Synthesis Assay Kit. Inhibition of protein clearance through the autophagy block produces HPG colocalization with the autophagosome marker LC3B (red), detected with the anti-LC3B antibody provided in the LC3B Antibody Kit for Autophagy. Cells were also stained with the Hoechst® 33342 nuclear counterstain (blue).

 

Product Quantity Cat. No.
Click-iT® HPG Alexa Fluor® 488 Protein Synthesis Assay Kit1 kitC10428
Premo™ Autophagy Sensor GFP-p62 Kit1 kitP36240
Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit1 kitP36239
LysoTracker® Deep Red Dye5 x 50 µLL12492

 

What's New With the BioProbes® Journal?

We are bringing our award-winning BioProbes® articles to you sooner. Instead of producing two print editions per year, 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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