ProbesOnline - July, 2011

  Read the latest issue of the ProbesOnline E-Newsletter.

In This Issue

FEATURED NEW PRODUCTS

 Easily Discriminate Apoptotic From Necrotic Cells — Tali™ Apoptosis Kit
   
 Proliferation Results Within 90 Minutes — Simplified Click-iT® EdU Kits for Flow Cytometry
   
 New Protein Disulfide Isomerase (PDI) Endoplasmic Reticulum Marker — ABfinity™ Recombinant Rabbit Monoclonal Antibodies

PROVEN PERFORMERS

 Multiparametric Cell Health Assays

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Molecular Probes Handbook
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FEATURED NEW PRODUCTS


Easily Discriminate Apoptotic From Necrotic Cells — Tali™ Apoptosis Kit

what it is
The Tali™ Apoptosis Kit contains two fluorescent reagents that discriminate apoptotic from necrotic cells—the green-fluorescent annexin V–Alexa Fluor® 488 conjugate, and red-fluorescent propidium iodide. Used with the new Tali™ Image-Based Cytometer, this kit enables the identification of apoptotic cells and the discrimination of apoptotic from necrotic cells in a population. 



what it offers

  • Contains all reagents for the fluorescent detection of apoptotic and necrotic cells
  • Quantitative image analysis of cell populations undergoing apoptosis
  • Optimized and ready to use with the Tali™ Image-Based Cytometer

how it works
In apoptotic cells, phosphatidylserine is translocated to the cell surface, where it can bind to the green-fluorescent annexin V–Alexa Fluor® 488 reagent. Propidium iodide is a cell-impermeant red fluorogenic dye that enters dead or necrotic cells and binds to DNA. After addition of the reagents and measurement with the Tali™ Image-Based Cytometer, cells are identified as apoptotic (green), dead (red or yellow), and live (no fluorescence).

Apoptosis assay with the Tali™ cytometer

Tali™ Apoptosis assay.


Jurkat cells were induced with 10 µM camptothecin. At selected time points, cells were stained using the Tali™ Apoptosis Kit and analyzed with the Tali™ Image-Based Cytometer. The sample tab (right) shows the concentration, relative proportion, and number of live, dead, and apoptotic cells. The image window (left) displays the captured fields of view, where the apoptotic cells with green fluorescence are clearly distinguishable from dead or necrotic cells that fluoresce red and live cells that do not fluoresce. Yellow-fluorescent cells are stained with both apoptotic and necrotic dyes, and are identified as dead.

Proliferation Results Within 90 Minutes — Simplified Click-iT® EdU Kits for Flow Cytometry

what they are
New streamlined kits for cell proliferation analysis using the Click-iT® EdU Flow Cytometry Assay are available. Standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® detection reagent to access DNA and label the incorporated EdU, making direct S-phase measurements available typically in less than 90 minutes.



what they offer

  • Streamlined step-by-step protocols
  • Two kit sizes: 50- and 100-test kits
  • Results typically obtained within 90 minutes

how they work
Incorporation of EdU provides a direct measurement of new DNA synthesis in cells, and eliminates the need to denature DNA for detection using click chemistry. The Click-iT® advantage is in the chemistry—small, unique, low-background labeling and detection moieties react specifically and covalently, giving consistent results time after time.

Dual parameter plot Click-iT EdU

Dual-parameter plot of Click-iT® EdU Alexa Fluor® 488 and FxCycle™ Violet fluorescence.
Product
Quantity
Cat. No.
Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit
50 assaysC10425Order Now
100 assaysC10420Order Now
Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit
50 assaysC10424Order Now
100 assaysC10419Order Now
Click-iT® EdU Pacific Blue™ Flow Cytometry Assay Kit
50 assaysC10418Order Now

New Protein Disulfide Isomerase (PDI) Endoplasmic Reticulum Marker — ABfinity™ Recombinant Rabbit Monoclonal Antibodies

what they are
Protein disulfide isomerase (PDI) is a resident protein of the endoplasmic reticulum (ER). PDI can be used as a marker for the ER, and has been implicated in many misfolded protein pathways.



what they offer
• Recombinant ABfinity™ antibodies enable consistent results
• New ABfinity™ antibodies released every month

how they work
ABfinity™ recombinant rabbit monoclonal antibodies help ensure consistent antibody performance lot after lot, so you don’t have to revalidate dilutions for your experiments when you order more. PDI ER marker antibodies are available as ABfinity™ Recombinant Rabbit Monoclonal Antibody and Recombinant Rabbit Oligoclonal Antibody preparations, and are validated for western blotting and ELISA applications. For more ER labeling and detection applications, we offer the SelectFX® Alexa Fluor® 488 ER Labeling Kit and CellLight® Endosomes-GFP and -RFP reagents.

Western blot analysis of whole cell extract using PDI ABfinity™ Recombinant Rabbit Monoclonal Antibody

Western blot analysis of whole cell extract using PDI ABfinity™ Recombinant Rabbit Monoclonal Antibody.


30 µg of protein from HEK cells was separated by SDS-PAGE, followed by transfer onto nitrocellulose. The blot was blocked and then incubated with PDI ABfinity™ Recombinant Rabbit Monoclonal Antibody at 2.5 µg/mL for 2 hr. A goat anti-rabbit HRP conjugate was used at 1:5,000 as the secondary antibody, and the blot was developed using chemiluminescence (ECL). The expected size of PDI is ~57 kDa.

PROVEN PERFORMERS


Multiparametric Cell Health Assays

Are my cells alive? Proliferating? How did my compound affect cell viability?

Is my compound cytotoxic? Researchers across many areas of cellular biology ask these questions when planning and performing experiments. Cell health (viability, proliferation, cytotoxicity, and cell death) is one of the more complex areas of cell biology, and answers can be provided by a wide variety of assays for various aspects of cell structure and function. Common cell health indicators measure reduction potential/metabolic status, membrane integrity, induction of proteases, loss of mitochondrial membrane potential, dsDNA breaks, nucleic acid content, and more.

No single assay can give a definitive answer on the health of a cell, and it is therefore common to use more than one approach when assessing cellular health at any given time point. Cell health assays inform important decisions in both academic and drug discovery research, and unambiguous answers are essential. To help answer such questions with more confidence, we have developed multiparametric cell health assays that offer:

  • Higher data quality—simultaneous detection of multiple parameters improves data quality and reduces risk of false negatives
  • Improved relevance—combination of cell health readouts (e.g., mitotoxicity, cytotoxicity, and morphology) enhances correlation to in vivo models
  • Cost and time savings—more than one readout in a single assay

 

 

Imaging of mitotoxicity and cytotoxicity of valinomycin in HeLa cells
 Imaging of mitotoxicity and cytotoxicity of valinomycin in HeLa cells using the HCS Mitochondrial Health Kit. HeLa cells were treated in a dose-response experiment with valinomycin between 2 nM and 120 μM final concentrations or with an equal volume of DMSO (control), incubated for 24 hr at 37°C/5% CO2, and assayed using the HCS Mitochondrial Health Kit.
The Thermo Scientific Cellomics® ArrayScan® VTI platform was used to obtain images of fixed cells at 20x. In the absence of valinomycin, the nucleus (detected with Hoechst 33342) and mitochondrial reticulum (detected with MitoHealth stain) were clearly visible, yet no signal due to permeability of plasma membranes (Image-iT® DEAD Green™ viability stain) was observed. At valinomycin concentrations in the nanomolar range, the mitochondrial reticulum was not observed as a defined structure, indicating valinomycin-induced loss of mitochondrial membrane potential. Loss of plasma membrane integrity did not occur at this level of valinomycin exposure, hence there was no fluorescence staining by the Image-iT® DEAD Green™ viability stain. At higher valinomycin concentrations (120 μM), however, plasma membrane integrity was compromised and resulted in intense nuclear fluorescence from the Image-iT® DEAD Green™ viability stain, indicating more overt cytotoxicity and cell death.

DEPARTMENTS


On the Web

Watch the Tali™ Cytometer in Action 

Watch the Tali™ Cytometer in Action

See a preview of the new Tali™ Image-Based Cytometer, a 3-channel benchtop assay platform that measures fluorescent protein expression, cell viability, and apoptosis.

The View

Mouse intestine thin section visualized with Alexa Fluor® 488 Phalloidin Conjugate
 
Mouse intestine thin section visualized with Alexa Fluor® 488 phalloidin conjugate, Cy®3-laminin, and DRAQ5. Image contributed by Michael W. Davidson, Florida State University.

Buzzworthy

Narita M, Young AR, Arakawa S et al. (2011) Science 332:966–970.

In a recent publication, Narita et al. propose a model by which cells can achieve both protein synthesis and autophagic degradation simultaneously. Using Premo™ Autophagy Sensor LC3B-RFP to investigate autophagy, CellLight® Golgi-GFP to localize and study the function of the Golgi complex, and Click-iT® Homopropargylglycine (HPG) to detect nascent protein synthesis, the researchers observed that, in cells undergoing senescence, autophagosomes fuse with mTORC1-containing lysosomes to form a specialized compartment (TASCC) for the execution of the secretory program. Because this compartment enforces a close proximity between the autophagy process and mTORC1, the amino acids and metabolites produced during autophagy activate mTORC1 to boost protein synthesis. These results demonstrate that cells can, under certain conditions, couple mass breakdown (catabolism via autophagy) to mass production (anabolism via protein synthesis) through the formation of a novel compartment, the TASCC.



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