ProbesOnline August 2011

  Read the latest issue of the ProbesOnline E-Newsletter.

In This Issue


Imaging Reagent Guide and Protocols at Your Fingertips   Imaging Reagents and Protocols at Your Fingertips — Cell Imaging App Now Available for the iPad®
 Quantitative Cell Viability Measurements at Your Benchtop   Quantitate Cell Viability at Your Benchtop — Tali™ Viability Kits
Unravel the Molecular Mechanisms of Insulin Resistance   Unravel the Molecular Mechanisms of Insulin Resistance — ABfinity™ Recombinant Rabbit Monoclonal Antibodies to AKT Substrates




Great Lakes International Imaging and Flow Cytometry Association
September 30–October 2
Olympia Resort and Conference Center
Oconomowoc, WI 



Imaging Reagents and Protocols at Your Fingertips — Cell Imaging App Now Available for the iPad®

what it is
With over 6,000 downloads, the Molecular Probes® Cell Imaging Reagent Guide & Protocols mobile app is designed to help you find fluorescent dyes, reagents, and protocols for fluorescence microscopy research applications. Now available for iPad®, iPhone®, iPod touch®, and Android™ devices.

what it offers

  • Productivity—easily find reagents and protocols in 20 application areas
  • Convenience—concise protocols with built-in timer and alarm
  • Simplicity—intuitive and easy to use

how it works
Get mobile access to protocols for labeling organelles and cellular structures, including the Golgi complex, mitochondria, nuclei, endoplasmic reticulum, plasma membrane, actin, tubulin, peroxisomes, autophagosomes, endosomes, and lysosomes. Also enjoy the convenience of a built-in protocol timer and alarm for timed steps.

Cell Imaging Mobile App for the iPad®

Cell Imaging Mobile App.

Quantitate Cell Viability at Your Benchtop — Tali™ Viability Kits

what they are
The Tali™ Viability Kits, optimized for the Tali™ Image-Based Cytometer, are used to quantitate cell viability in minutes, right at your benchtop. Results are comparable to those obtained with a flow cytometer.

what they offer

  • Streamlined workflow—ready-to-use and optimized for the Tali™ Image-Based Cytometer
  • Dual-parameter fluorescence analysis—the Dead Cell Green and Dead Cell Red reagents can be used in conjunction with RFP and GFP, respectively
  • Fast and easy cell viability determination—quantitative results generated typically in less than 5 minutes

how they work
The quantitative measurement of cell viability is an important consideration for any laboratory performing cell culture and/or cellular assays. The Tali™ Viability Kit reagents are composed of fluorescent dyes—propidium iodide (Dead Cell Red Kit) and SYTOX® Blue (Dead Cell Green Kit)—that readily enter necrotic cells and bind to nucleic acids. These dyes have been optimized for use with the fluorescence channels on the Tali™ instrument. The use of a single fluorescence channel leaves the other channel available for the detection of other fluorescent dyes or proteins.

The Tali™ Viability Kit

The Tali™ Viability Kit compared to flow cytometry.

( A) The percentage of viable cells from the total population was measured with five different cell types. ( B) Time-course comparison of cell viability. 10% (v/v) ethanol was added to 293MSR cells to induce cell death. Population data measured with the Tali™ Image-Based Cytometer are comparable to those obtained using a flow cytometer.

Unravel the Molecular Mechanisms of Insulin Resistance — ABfinity™ Recombinant Rabbit Monoclonal Antibodies for AKT Substrates

what they are
The AKT substrates AS160 and PRAS40 have been observed to accompany insulin signaling and glucose disposal in type 2 diabetes. Investigating the balance between glucose and signaling proteins can provide insight into the mechanisms of diabetes.

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. AS160 phosphothreonine 642 ([pT642]) and PRAS40 antibodies are available as ABfinity™ Recombinant Rabbit Monoclonal and Oligoclonal Antibody preparations, and are validated for western blotting and ELISA applications.

Western blot analysis of whole cell extract using AS160 [pT642] ABfinity™ Recombinant Rabbit Monoclonal Antibody

Western blot analysis of whole-cell extract using AS160 [pT642] ABfinity™ Recombinant Rabbit Monoclonal Antibody.

Whole-cell extract (80 µg per lane) from serum-starved HeLa cells treated with IGF-1 (150 ng/mL for 15 min) was separated by SDS-PAGE and transferred to nitrocellulose. The blot was blocked and incubated with 2.5 µg/mL AS160 [pT642] ABfinity™ Recombinant Rabbit Monoclonal Antibody, overnight at 4°C. HRP-conjugated goat anti-rabbit secondary antibody was used at a 1:5,000 dilution, and the blot was developed using chemiluminescence (ECL). A  band at ~160 kDa, corresponding to AS160, was detected.


Spatiotemporal Imaging of Protein Synthesis and Autophagy

The CellLight® reagents and Premo™ Autophagy Sensors combine the utility and selectivity of fluorescent proteins with the transduction efficiency of BacMam technology, enabling unambiguous visualization of organelles, cellular structures, and processes in live mammalian cells by fluorescence microscopy. Provided in ready-to-use format—simply add, incubate, and visualize—these reagents open new avenues for multiparametric studies of dynamic cellular events. Unlike expression vectors, BacMam reagents allow controlled and reproducible transient expression.

CellLight® and Premo™ Autophagy BacMam 2.0 reagents provide several advantages over other gene transfer methods:

  • Efficient—>90% transduction efficiency in a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
  • Fast and convenient—simply add the BacMam 2.0 reagent and cells to your coverslip or microtiter plate, incubate overnight, and assay; or store frozen, assay-ready cells for later use
  • Safe—nonreplicating in mammalian cells, lack of observable cytopathic effects
  • Flexible—cotransduce multiple BacMam reagents for multiplex experiments or colocalization studies, and tightly control expression levels by just varying the dose

The combination of these reagents with more traditional imaging tools has been illustrated in several recent elegant studies. Narita et al. proposed a mechanism for the mTOR-dependent coregulation of protein synthesis and autophagic degradation during Ras-induced senescence. The authors revealed a self-enhancing system enabled by close spatial integration of rER-Golgi, autolysosomes, and mTOR, in which autolysosome-derived amino acids reinforce mTOR enrichment and activity. The authors suggested that this “TOR-autophagy spatial coupling compartment” (TASCC) may represent a general mechanism for rapid protein turnover. In addition to employing Premo™ Autophagy Sensor LC3B-RFP and CellLight® Golgi-GFP for colocalization studies, the authors used Click-iT® reagents based on “clickable” amino acid analogs (Click-iT® HPG and Click-iT® AHA) to visualize nascent proteins.


Visualization of nascent protein synthesis in Ras-induced senescent IMR90 cells
Visualization of nascent protein synthesis in Ras-induced senescent IMR90 cells.
Cells were precultured for 30 min in methionine-free medium before metabolic labeling with a reactive methionine analog, Click-iT® L-homopropargylglycine (HPG). After a 30 min labeling with HPG, cells were fixed and stained for HPG and TGN46 (a marker of the trans-Golgi network, TGN). Nascent protein was accumulated in the TGN. Image used with permission from Masako Narita, Cambridge Research Institute.



Copper-Free Click-iT® DIBO Reagents Preserve Cell Viability and Protein Function

  • Gentle reaction—no copper needed as catalyst
  • Bio-orthogonal—no side reactions with biological components
  • Compatible with live cells
  • Compatible with fluorescent proteins
  • Compatible with fluorescent phalloidin

Dibenzocyclooctyne (DIBO) compounds react with azide-modified macromolecules to form a stable, covalent 1,2,3-triazole linkage in the absence of copper, enabling the study of live cells without copper toxicity. The use of DIBO compounds also allows simultaneous imaging with fluorescent proteins, which can be quenched in the presence of high copper concentrations. Particularly suited for imaging applications are the Click-iT® DIBO Alexa Fluor® compounds. Additionally, a DIBO handle can be introduced into proteins and other macromolecules with reactive probes capable of modifying amines, thiols, and carboxy groups. The Click-iT® biotin DIBO derivative is a means of introducing biotin groups at any azide-modified position in a biological molecule, allowing detection by microscopy and flow cytometry, and enrichment by techniques involving streptavidin or avidin.



Live-cell labeling of surface glycoproteins with Click-iT® Alexa Fluor® compounds
click to enlarge
  Live-cell labeling of surface glycoproteins with Click-iT® Alexa Fluor® DIBO compounds. HeLa cells were metabolically labeled for 3 days with tetraacetylated N-azidoacetyl-D-mannosamine (ManNAz), which introduces the N-azidoacetyl analog of sialic acid (SiaNAz) into cell surface and secreted glycoproteins. Click-iT® DIBO-Alexa Fluor® 488, Click-iT® DIBO-Alexa Fluor® 555, Click-iT® DIBO-Alexa Fluor® 594, or Click-iT® DIBO-Alexa Fluor® 647 was used to detect the cell surface labeled sialoglycoproteins. The nuclei were visualized with Hoechst 33342. Cells were imaged after fixation with 4% formaldehyde. Images were collected with the Thermo Scientific Cellomics ArrayScan® VTI platform. Top panels: control cells not treated with ManNAz; bottom panels: cells treated with ManNAz.



From the Bench

Targeted Identification of Metastasis-Associated Cell-Surface Sialoglycoproteins in Prostate Cancer

Yang L, Nyalwidhe JO, Guo S, Drake RR, Semmes OJ (2011) Mol Cell Proteomics 10(6):M110.007294.

Yang et al. recently described a method using Click-iT® ManNAz metabolic glycoprotein labeling reagent (Ac4ManNAz) for the metabolic labeling, enrichment, and identification of sialoglycoproteins in nonmetastatic vs. metastatic syngenic prostate cancer cell lines. The cultured cells were fed Ac4ManNAz for 1–3 days, and to confirm metabolic labeling of cell-surface glycoproteins, the cells were fixed and click-labeled with the Click-iT® Biotin Protein Analysis Detection Kit and analyzed by confocal microscopy and flow cytometry. After confirmation of labeling, the azide-labeled cellular lysates were reacted with biotin-alkyne, and the labeled sialoglycoproteins were enriched on streptavidin resin. The resin-bound glycoprotein fraction was eluted and analyzed by SDS-PAGE followed by liquid chromatography–tandem MS. The results showed that the Click-iT® assay–based enrichment approach gave, on average, a 3-fold increase in the number of cell surface sialylated glycoproteins identified. The results also showed that multiple sialoglycoproteins are overexpressed in the metastatic cell lines and that most of these proteins are known to be involved in cell motility, migration, and invasion.

On the Web

ProLong® Fold Antifade Reagent   Our antifade kits and reagents have been shown to increase the photostability of, and to reduce initial quenching of, many of our fluorophores in fixed cells, fixed tissues, and cell-free preparations.

Imaging Corner

Wound healing in neonatal dermal fibroblasts
  Wound healing in neonatal dermal fibroblasts.
(Left) 24 hours post-wound, treated with vehicle. (Right) 24 hours post-wound, treated with 0.1 µM cytochalasin D. Gibco® human neonatal dermal fibroblasts were stained with Alexa Fluor® 488 phalloidin, anti-tubulin IgG, and goat-anti mouse Alexa Fluor® 555 conjugate.

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