ProbesOnline September 2011

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In This Issue

FEATURED NEW PRODUCTS


Attune® Acoustic Focusing Cytometer—Now Available in Blue/Red Laser Configuration

what it is
With the Attune® Acoustic Focusing Cytometer, you can get the best of both worlds: high sample-throughput rate and high sensitivity for analyzing precious samples or detecting rare events. The Attune® cytometer is now available with a blue/red laser configuration, so you can select the configuration that best fits your research needs.



what it offers

  • Precision—high collection rates without compromising CV
  • Speed—rare-event detection up to 10 times faster than other cytometers
  • Simplicity—no-lyse, no-wash method avoids cell loss

how it works
Using the Attune® cytometer with the red laser option, analysis of stained mouse whole blood with a stain/lyse protocol shows excellent separation of cell populations into subsets for immunophenotyping experiments using up to 6 colors. There is strong signal separation for data clarity, and 6-color detection is easily performed with the automated compensation module. In addition, the Attune® cytometer generates less waste than other systems do, due to its low sheath fluid usage per run.

Attune® Acoustic Focusing Cytometer

Six-color immunophenotyping analysis on the Attune® Acoustic Focusing Cytometer with red laser option.
 

Mouse (C57BL/6J) peripheral blood is stained with a 6-color immunophenotyping panel of anti-mouse direct dye conjugates: CD3, hamster anti-mouse (Alexa Fluor® 647 dye); CD19, rat anti-mouse (PE-Cy®5.5 dye); CD4 PE-Cy®7 dye; and CD8a, rat anti-mouse (Alexa Fluor® 488 dye). Gating was performed on lymphocytes for each of the bivariate plots. The plots show a 2-color representation of the 6-color panel identifying the major T and B cell subsets of the lymphocyte populations.
Product
Quantity
Cat. No. To Order
Attune® Acoustic Focusing Cytometer (blue/violet configuration, includes computer, monitor, startup solutions, installation, and warranty)
1 each4445315Contact your Sales Representative
Attune® Acoustic Focusing Cytometer (blue/red configuration, includes computer, monitor, startup solutions, installation, and warranty) 
1 each4469120Contact your Sales Representative

Measure Interferon-γ Reliably Using the IFN-γ Mouse ELISA Kit

what it is
The Invitrogen™ IFN-γ Mouse ELISA Kit offers accurate quantitation of IFN-γ in mouse serum, plasma, or tissue culture supernatant samples. IFN-γ plays an important role in innate and adaptive immunity and exhibits both immunostimulatory and immunomodulatory effects. IFN-γ has antiviral as well as antitumor properties, and differs from IFN-α and IFN-β subunits in that it is only secreted by activated T and NK cells.



what it offers

  • Easy to run
  • Convenient precoated, removable 8-well strips
  • Sensitivity of <2pg/mL

how it works
The kit includes reagents for running conventional sandwich ELISAs in approximately 4 hours. Samples are incubated in 96-well plates precoated with IFN-γ antibody. A biotin-conjugated secondary antibody is then added, followed by detection with streptavidin-HRP and a stabilized chromogenic substrate. Quantitative results are obtained using a microplate reader.

IFN-γ Mouse ELISA Kit

Parallel results with natural and recombinant mouse IFN-γ.


The Invitrogen™ IFN-γ Mouse ELISA Kit provides analytical sensitivity of <2 pg/mL and a dynamic range of 4.7–300 pg/mL target protein. Data show that the recombinant standard provided in the kit gives values parallel to those from a tissue culture supernatant sample.

Simple, Cost-Effective Immunoassay Multiplexing—The New MAGPIX® System

what it is
The MAGPIX® system is a versatile, affordable, and compact fluorescence detection system based on proven Luminex® xMAP® bead-based technology. The MAGPIX® system is capable of performing up to 50 multiplex immunoassays on a single biological sample. It helps deliver fast, accurate, and reproducible results while saving time and cost and conserving valuable samples.



what it offers

  • Ideal immunoassay tool—simultaneous analysis of up to 50 proteins from a limited volume of precious samples
  • Excellent performance—accurate, reproducible, and sensitive quantification of proteins
  • Reduced time and cost—compared to multiple- and single-analyte ELISAs or western blots
  • Easy to use—designed to work with a broad and expanding range of Invitrogen™ immunoassay magnetic bead kits

how it works
The MAGPIX® system captures data from singleplex and multiplex immunoassays. It is ideal for both new and experienced Luminex® technology users. The MAGPIX® instrument makes use of novel light-emitting diodes (LEDs) for excitation and CCD camera technology for bead and analyte detection. As a result, the MAGPIX® system is inexpensive and occupies minimal bench space. The system also has streamlined startup and shutdown protocols and requires minimal maintenance, making it easy to operate.

 Request information

The MAGPIX® System

The MAGPIX® System.
This compact and affordable Luminex® system performs up 50 assays simultaneously in a single well of a 96-well plate.

NEW APPLICATIONS


Zenon® Labeling Kits for Epigenetic Analysis of Brain Cells

  • Facilitates rapid (10 min), one-step labeling of nuclei that is stable enough for cell sorting
  • Low background—comparable to that associated with primary/secondary antibody approaches
  • Ideal for labeling delicate preparations

Zenon® labeling kits were designed for simple, rapid, and noncovalent labeling of the Fc portion of primary antibodies. Labeling reactions accommodate 1–20 µg of antibody, with no purification step required.

Recently Zenon® technology was used to label an antibody that recognizes an antigen present only on neuronal cells derived from brain tissue. The researchers used the labeled antibodies to separate neuronal from non-neuronal nuclei from human prefontal cortex cells using cell sorting. They then extracted DNA from the separated nuclei and evaluated the methylation patterns in each group (Genome Res 21:688 (2011)). The authors state, “Unraveling the epigenetic status of neuronal cells would, therefore, be critical to understand the molecular basis of brain functions.”
When labeling delicate samples, such as isolated prefrontal cortex nuclei, speed and labeling specificity are critical factors. Antibody labeling using Zenon® labeling technology is ideal for these types of investigations.

 

Separation of neuronal and non-neuronal nuclei by FACS
 
Separation of neuronal and non-neuronal nuclei by FACS.
(A) Typical example of nuclei sorting based on Alexa Fluor® 488–conjugated anti-NeuN antibody. (B) Microscopic examination of isolated neuronal (NeuN+) and non-neuronal (NeuN) nuclei. Note that the image did not reflect the yield because of the different levels of dilution. Figure and caption reproduced with permission from Genome Research, a publication of Cold Spring Harbor Press.

PROVEN PERFORMERS


Tracking Hematopoietic Stem Cells and Human Prostate Cancer Cells With Qtracker® Cell Labeling Kits and CellTracker™ Dyes

Qtracker® Call Labeling Kits

  • CellTracker™ and Qtracker® reagents offer intense, stable fluorescence that can be traced through several cell generations
  • Available in many colors for multiplexing with other reagents and with each other
  • Excellent for long-term tracking or imaging studies of live cells, including assays for migration, motility, morphology, and other cell functions
  • CellTracker™ and Qtracker® reagents tolerate the harsh fixation and mounting treatments needed for tissue staining


Qtracker® Cell Labeling Kits offer intense, stable fluorescent cell labeling that can be traced through several generations and is not transferred to adjacent cells in a population. Qtracker® kits are are available with Qdot® nanocrystals in seven brilliant colors—emitting at 525 nm, 565 nm, 585 nm, 605 nm, 655 nm, 705 nm, or 800 nm—and they are excellent for long-term studies of live cells and tissues, including assays for migration, motility, morphology, and other cell functions. In rapidly dividing cells, the Qdot® nanocrystals are distributed among the daughter cells, lowering the signal, but recent publications suggest that Qdot® nanocrystals can be tracked for up to 8 days, through more than 7 cell divisions [1]. Qdot® nanocrystals are also known for their superior resistance to the chemicals used for fixing, mounting, and staining tissue during in vivo studies [2]. These superior properties make Qtracker® kits perfect for tracking a variety of cells both in vivo and in vitro.

In a recent study, Yusuke Shiozawa et al. employed Qtracker® reagents for in vivo studies, and CellTracker™ reagents for in vitro analysis, in their research on prostate cancer [3]. They report that human prostate cancer cells target hematopoietic stem cells (HCSs) to establish a foothold in mouse bone marrow. Their data suggest that HSCs serve as a specific site (or niche) in which displaced prostate cancer cells can gain a foothold. These cancer cells also seem to compete against HCSs, or may be even mobilizing HCSs in bone marrow.

Table 1 lists other recent publications in which researchers used Qtracker® kits to track cells and cell lines in many different applications.

  1. Brown MR et al. (2010) Cytometry 77:925–932.
  2. Muccioli M et al. (2011) J Vis Exp 52 (2011)  doi: 10.3791/2785.
  3. Shiozawa et al. (2011) J Clin Invest 121(4):1298–1312.

 

Table 1. Research use of Qtracker® kits to track cells and cell lines.

Publication Utilization of Qtracker® Cell Labeling Kits
Isherwood et al. (2011) Pharmaceutics 3:141–170Demonstrated the usability of Qtracker® reagents in live cell imaging, especially for in vivo studies.
Boehm et al. (2011) AIP Advances 1:022139 Used Qtracker® reagents to modify microneedles using inkjet printing, which successfully delivered Qdot® nanocrystals to porcine skin. 
Jesuraja et al. (2011) J Neurosci Methods 197:209–215 Used Qtracker® reagents to track Schwann cells nerve grafts.
Ji Hye Seo et al. (2011) Nanotechnology 22: 235101 Loaded HeLa cells with Qtracker® reagents and investigated concentration-dependent intracellular movements of the Qdot® nanocrystals in real time.
Kim et al. (2011) Blood 117:3343–3352Tracked polymorphonuclear neutrophils in mouse wounds with Qtracker® reagents.
Mineharu et al. (2011) Clin Cancer Res 17:4705–4718Used Qtracker® reagents to stain dendritic cells (DC) in research aimed at evaluating antitumor immune response after dendritic cell (DC) vaccination in combination with other treatments.
Ranjbarvaziria et al. (2011) Biomaterials 32:5195–5205 Used different Qtracker® reagents (525, 585, and 800 nm) to label CD133, CD34, CD14, and mesenchymal stem cells. They reported excellent internalization and almost no effect on viability of cells. 
Rees et al. (2011) BMC Syst Biol 5:31Used Qtracker® reagents to determine the ratio of endosomes inherited by the two daughter cells during mitosis in the U2-OS human osteosarcoma cell line.
Summers et al. (2011) Nat Nanotechnol 6:170Did statistical analyses of dosing of Qtracker® reagents in U2-OS cells.
Zheng L et al. (2011) PLoS One 6(4): e19390Tracked Panc02 cells, labeled using the Qtracker® 565 kit, injected into the mouse splenic bed to show that tyrosine 23 phosphorylation–dependent cell-surface localization of annexin A2 is required for invasion and metastasis of pancreatic cancer.
Product Cat. No. Excitation (nm)Emission (nm)
Qtracker® 565 Cell Labeling KitQ25031MP405–525565
Qtracker® 625 Cell Labeling KitA10198405–585625
Qtracker® 655 Cell Labeling KitQ25021MP405–615655
Qtracker® 800 Cell Labeling KitQ25071MP405–760800
CellTracker™ Blue CMACC2110353466
CellTracker™ Green CMFDAC2925492 *517 *
CellTracker™ Orange CMTMR C2927541565
CellTracker™ Red CMTPXC34552577602
* CMFDA is colorless and nonfluorescent until the acetate groups are cleaved by intracellular esterases; hydrolysis of the acetates yields a product with the indicated spectral properties.

DEPARTMENTS


On the Web

Antibody Labeling Instructional Video Labeling a Purified Monoclonal Antibody?
We’ve posted an educational video on how to label your next antibody for imaging or fluorescence analysis. Walk through the tutorial as Molecular Probes scientists demonstrate the protocol—including all the tips and tricks you’ll want to know about for your next antibody-labeling experiment. The video features Judie showing Curtis, a chemistry graduate student, how to label his antibodies while saving time and maximizing yield.

Imaging Corner

Cell Division and Proliferation in Human Dermal Fibroblasts
 Cell Division and Proliferation in Human Dermal Fibroblasts
Gibco® Human Dermal Fibroblasts, Neonatal (HDFn) were treated with the nucleoside analog Click-iT® EdU. Cells were then fixed and permeabilized, and EdU that had been incorporated into newly synthesized DNA was detected with green-fluorescent labeling using the Click-iT® EdU Alexa Fluor® 488 Imaging Kit. Tubulin was labeled with a mouse anti-tubulin antibody and visualized with an Alexa Fluor® 555 goat anti–mouse IgG antibody. Nuclei were labeled with blue-fluorescent Hoechst 33342 nucleic acid stain. Image contributed by Nicholas Dolman, Life Technologies Corporation.

From the Bench

Inhibition of Ebola Virus Entry by a C-Peptide Targeted to Endosomes
Miller EH, Harrison JS, Radoshitzky SR et al. (2011) J Biol Chem 286:15854–15861.


Members of the filovirus family (Ebola virus and Marburg virus) are causative agents of severe and highly fatal diseases that have captured the imagination of the general public—graphic images of hemorrhagic fever symptoms generated by these agents are as horrific as they are gruesome. Fortunately, scientists are focusing efforts on developing treatments to inhibit infection and disease following infection with these viruses. In a recent article, scientists at the Albert Einstein College of Medicine and collaborators describe a study of peptides intended to inhibit virus entry into cells and thereby halt infection. Many enveloped viruses escape into the cytoplasm by membrane fusion following endosomal entry. In this study, a synthetic peptide that mimicked the fusion-active region of a viral-membrane protein was shown to inhibit membrane fusion and thus halt virus entry into cells by competing with endocytosed virus particles for the cellular fusion partner. The efficacy of the peptide was enhanced by fusion to an endosome-targeting peptide. Mechanistic and colocalization studies indicated that the antiviral peptide acted on an intermediate in the entry pathway. This study has thus confirmed the Ebola virus entry pathway and may contribute to development of therapeutic agents against this deadly disease. CellLight® Lysosomes-RFP BacMam 2.0 was used as a lysosomal colocalization marker in the study.


For Research Use Only. Caution: Not intended for human or animal therapeutic or diagnostic use.
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