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In This Issue

FEATURED NEW PRODUCTS

SYTOX® Dead Cell Stains Distinguish Dead from Live Cells in Flow Cytometry Assays — SYTOX® Dead Cell Stains
   
ABfinity™ Recombinant Monoclonal Antibodies for JAK-STAT Targets Identify Downstream Interferon Signaling Events — ABfinity™ Recombinant Monoclonal Antibodies for JAK-STAT Targets


NEW APPLICATIONS

Visualize Endocytosis With Transferrin, LDL, and Lipid Dyes Visualizing Endocytosis


PROVEN PERFORMERS

Alexa Fluor-labeled IgM Application Notes for the Countess® Automated Cell Counter

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UPCOMING MEETINGS

Northwest Flow Cytometry Meeting
March 24–26
Oregon Health & Science University
Portland, Oregon
Table #4

OTHER PUBLICATIONS

BioProbes 63
BioProbes®
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Expanded Content Now Online
Molecular Probes Handbook
The Molecular Probes® Handbook
11th Edition

FEATURED NEW PRODUCTS


Distinguish Dead from Live Cells in Flow Cytometry Assays — SYTOX® Dead Cell Stains

what they are
SYTOX® Dead Cell Stains are bright, easy-to-use cell-impermeant nucleic acid stains for distinguishing dead from live cells. The newly formulated SYTOX® Green Dead Cell Stain, SYTOX® Orange Dead Cell Stain, and SYTOX® Dead Cell Stain Sampler Kit are optimized for flow cytometry.



what they offer
  • Validated—dye concentration is optimized for use in flow cytometry
  • Bright signal—easily identify dead cells from live cells
  • Rapid staining—single-step, no-wash assay
how they work
Because SYTOX® dyes do not cross intact cell membranes and do increase fluorescence upon dsDNA binding, they are among our most brilliant dead cell stains. Dead cells can be easily distinguished from live cells by flow cytometry by measuring the increased fluorescence signal due to dsDNA binding. SYTOX® Dead Cell Stains can be applied to cells and visualized without an additional wash step because they are nonfluorescent in aqueous media.

SYTOX® Dead Cell Stains

Dead cell discrimination using SYTOX® Green Dead Cell Stain.


A mixture of heat-killed and live Jurkat cells was stained with 30 nM SYTOX® Green Dead Cell Stain and incubated at room temperature for 20 min. Cells were analyzed on the Attune® Acoustic Focusing Cytometer equipped with a 488 nm laser. Fluorescence emission was collected using a 530/30 bandpass filter. Live cells are easily distinguished from the brighter dead cell population.
Dye Excitation Source Excitation MaximumEmission Maximum Cat. No.  
SYTOX® Green Dead Cell Stain for flow cytometry488 nm504 nm523 nmS34860Order Now
SYTOX® Orange Dead Cell Stain for flow cytometry488, 532 nm547 nm570 nmS34861Order Now
SYTOX® Dead Cell Stain Sampler KitVariousVariousVariousS34862Order Now

Identify Downstream Interferon Signaling Events — ABfinity™ Recombinant Monoclonal Antibodies for JAK-STAT Targets

what they are
ABfinity™ recombinant rabbit monoclonal antibodies are available for many targets, including JAK1 [phospho Y1022], STAT1 [phospho Y701], STAT4, and STAT6 [phospho Y641]. JAK-STAT signaling is important in regulating immune responses and autoimmune diseases, especially when interferon-γ signaling is compromised in various viral infections.



what they offer

  • High sensitivity and specificity
  • Excellent lot-to-lot consistency
  • Extensive validation and characterization

 

how they work
ABfinity™ recombinant rabbit monoclonal antibodies are produced from specific recombinant clones. ABfinity™ technology helps to ensure consistent antibody performance so you don’t have to reoptimize your assay with each new lot.

Phosphorylated STAT1 (Y701) detected in interferon-γ–stimulated or unstimulated HeLa cells.

Phosphorylated STAT1 (Y701) detected in interferon-γ–stimulated HeLa cells.


Cells were stimulated with 100 ng/mL interferon-γ (top right) or unstimulated (top left) and labeled with rabbit anti-STAT1 [pY701] (1 µg/mL). Stimulated cells were preincubated with the phosphopeptide used as an immunogen (bottom right) or with nonphosphopeptide (bottom left), before incubating with Alexa Fluor® 488 goat anti–rabbit IgG secondary antibody at 1:1,000 dilution. The signal is prevented by incubation with the phosphopeptide but not with the nonphosphopeptide, demonstrating phosphospecifity of the antibody.
Product
Species Quantity
Cat. No.
STAT1 [pY701] ABfinity™ Recombinant Rabbit Monoclonal Antibody, PurifiedHuman
100 µg
700349Order Now
STAT4 ABfinity™ Recombinant Rabbit Monoclonal Antibody, Purified
Human100 µg700185Order Now
STAT6 [pY641] ABfinity™ Recombinant Rabbit Monoclonal Antibody, PurifiedHuman100 µg700247Order Now
JAK1 [pY1022/pY1023] ABfinity™ Recombinant Rabbit Monoclonal Antibody, Purified
Human100 µg700028Order Now

NEW APPLICATIONS


Visualizing Endocytosis

Lysosomally directed and recycling endosomal pathways are critical for a variety of cellular functions. Labeled and unlabeled low-density lipoprotein (LDL), transferrin, lipid dyes such as FM® lipophilic styryl dyes and lipophilic tracers (Dil, DiO, DiD, DiA, and DiR), and pHrodo™ indicators are valuable tools for interrogating endocytic pathways. In a recent study, Boucrot et al. [1] used several of these tools to determine the importance of the AP2 protein in the mechanism of endocytosis.

Transferrin Conjugates
Transferrin is a monomeric serum glycoprotein that binds up to two Fe3+ atoms for delivery to vertebrate cells through receptor-mediated endocytosis. Fluorescently labeled transferrin has been used to measure transferrin receptor-binding affinity in mammals and parasites, to investigate endocytosis, and to study endocytic recycling pathways. Alexa Fluor® transferrin conjugates are highly recommended because of their brightness, enhanced photostability, and lack of sensitivity to pH.

Fluorescent Low-Density Lipoprotein Complexes
The human LDL complex delivers cholesterol to cells by receptor-mediated endocytosis. In addition to unlabeled LDL, which has been reported to be an effective vehicle for selectively delivering antitumor drugs to cancer cells, we offer two classes of labeled LDL probes—those containing an unmodified apoprotein, used to study the mechanisms of normal cholesterol delivery and internalization, and those with an acetylated apoprotein, used to study endothelial, microglial, and other cell types that express receptors that specifically bind this modified LDL.

Lipid Dyes
Lipid dyes are useful as general-purpose probes for investigating endocytosis and for simply identifying cell membrane boundaries. We offer FM® lipophilic styryl dyes and lipophilic tracers (Dil, DiO, DiD, DiA, and DiR), which fluoresce brightly upon integration into cellular membranes. Both fixable and unfixable dyes are available in various colors.

pHrodo™ Indicators

The pH-sensitive pHrodo™ dye is nonfluorescent at neutral pH and fluoresces bright red in acidic environments. This increase in fluorescence signal at low pH makes it ideal for studying endocytosis and its regulation by drugs and/or environmental factors. The unique pHrodo™ dye–based system measures phagocytosis and endocytosis based on acidification of the particle or protein conjugates as they are ingested by cells.  

1. Boucrot E, Saffarian S, Zhang R et al. (2010) Roles of AP-2 in Clathrin-Mediated Endocytosis. PLoS ONE 5(5): e10597.




Visualizing transferrin and transferrin receptors.
 
Visualizing transferrin and transferrin receptors.
A431 cells were incubated with green-fluorescent Alexa Fluor® 488 transferrin, then fixed and permeabilized. Transferrin receptors were identified with anti–transferrin receptor, mouse IgG1 monoclonal antibody and visualized with red-fluorescent Alexa Fluor® 555 goat anti–mouse IgG antibody. Yellow fluorescence indicates regions of colocalization. Nuclei were stained with DAPI.

Product
Quantity
Cat. No.
Low-density lipoprotein from human plasma (LDL), 2.5 mg/mL
200 μL
L3486Order Now
Low-density lipoprotein from human plasma, acetylated, Alexa Fluor® 488 conjugate (Alexa Fluor® 488 AcLDL)
200 μL
L23380Order Now
Low-density lipoprotein from human plasma, acetylated, Alexa Fluor® 594 conjugate
200 μL
L35353Order Now
Transferrin from human serum, Alexa Fluor® 594 conjugate
5 mgT13343Order Now
Transferrin from human serum, Alexa Fluor® 488 conjugate
5 mg
T13342Order Now
FM® 1-43FX, fixable analog of FM® 1-43 membrane stain
10 x 100 μg
F35355Order Now
DiOC18 (3,3′-dioctadecyloxacarbocyanine perchlorate)
100 mg
D275Order Now
Dextran, pHrodo™ 10,000 MW for endocytosis
0.5 mg
P10361Order Now

PROVEN PERFORMERS


Application Notes for the Countess® Automated Cell Counter


Countess® Automated Cell Counter The Countess® Automated Cell Counter counts live and dead cells and measures viability, typically in just 30 seconds. For specific protocols and technical information, you can download a variety of application notes relating to your research area. Topics include:

  • Counting clumpy primary cells
  • Counting blood cells
  • Reducing trypan blue concentration
  • Counting stem cells
  • Improving downstream results

Product
Quantity
Cat. No.
Countess® Automated Cell Counter
1 each
C10227Order Now
Countess® Automated Cell Counter Starter Kit with 11 boxes of slides
1 kitC10310Order Now
Countess® Automated Cell Counter Starter Kit with 101 boxes of slides
1 kitC10311Order Now

DEPARTMENTS


Buzzworthy

SYTO® Probes in the Cytometry of Tumor Cell Death
Wlodkowic D, Skommer J, Darzynkiewicz D (2008) Cytometry Part A 73A:496–507.

High-throughput screening platforms to analyze anticancer treatments and other therapies are valuable tools in the modern clinical research setting. A recent publication from Wlodkowic and colleagues surveys a few of the patented DNA-binding SYTO® dyes for the detection and tracking of apoptosis in diverse cell lines and in primary tumor samples. Loss of apoptosis is one of the defining parameters of malignancy, and the mode of action of some of the SYTO® dyes makes them particularly suited for monitoring this cellular process. Wlodkowic et al. cite the broad range of visible excitation and emission, high quantum yield upon binding to nucleic acids, low cost, and low toxicity profiles as beneficial in their investigations. They speculate that these probes will be useful as a first-line screening agent for novel therapeutics and that they could be very valuable in multiplexed assays and in microfluidics devices.

The View

Transverse Section of Fixed Zebrafish Retina.
 Transverse Section of Fixed Zebrafish Retina. A transverse section of fixed zebrafish retina was probed with FRet 43, a monoclonal antibody that binds to double cone cells, and visualized using the ELF® 97 Immunohistochemistry Kit.

The yellow-green–fluorescent double cones stained with the ELF® 97 alcohol precipitate are oriented with the outer segments at the top of the stained configuration and the synaptic pedicles at the bottom. This section was counterstained with tetramethylrhodamine wheat germ agglutinin, which makes the rod outer segments (top left) and the inner plexiform layer and ganglion cell axons (bottom right) appear bright red. Wheat germ agglutinin also binds the region occupied by the cone outer segments and synaptic pedicles, which appear bright yellow because they are double-labeled with both the ELF® 97 alcohol precipitate and tetramethylrhodamine. Although this section has also been counterstained with Hoechst 33342, the blue-stained nuclei are barely visible in this photomicrograph. However, the double cones' inner fibers traverse the region occupied by the rod nuclei, and thus appear light blue. The inner segments, myoids, and nuclei of these double cone cells are labeled only with the ELF® 97 alcohol precipitate, giving them a characteristic green appearance. The image was obtained by triple exposure through optical filters appropriate for DAPI, tetramethylrhodamine, and the ELF® precipitate. Used with permission from J Histochem Cytochem 43:77 (1995).

 

Product Quantity Cat. No.  
ELF® 97 Immunohistochemistry Kit1 kitE6600Order Now
Hoechst 33342, trihydrochloride, trihydrate100 mgH1399Order Now
Hoechst 33342, trihydrochloride, trihydrate, FluoroPure™ grade100 mgH21492Order Now
Hoechst 33342, trihydrochloride, trihydrate, 10 mg/mL solution in water10 mLH3570Order Now
Wheat germ agglutinin, tetramethylrhodamine conjugate5 mgW849Order Now

What's New

Attune™ Acoustic Focusing Cytometer Attune® Cytometer Named One of Top 10 Innovations in 2010

The Scientist named the Applied Biosystems® Attune® Acoustic Focusing Cytometer one of the top 10 innovations of 2010. Here’s what the judges said:

“Designed to use sound waves to precisely control the movement of cells and increase instrument simplicity, sensitivity and throughput. Looks like it will be particularly useful for analyzing dilute cell samples. The simplicity and relatively low cost of the instrument should also increase the number of scientists who use flow cytometry.”
H. Steven Wiley, Lead Biologist, Pacific NW National Labs (USA)

“With a footprint small enough to fit in a laminar-flow hood and a completely new approach to fluidics, the Attune cytometer promises less clogging than other flow cytometers, even while speeding through huge populations of cells.”
Amy A. Caudy, Research Fellow, Lewis-Sigler Inst. for Integrative Genomics, Princeton University

On the Web

Immunoassay Selection Guide Immunoassay Selection Guide

Finding the right ELISA kit, Luminex® assay, or antibody pair just got easier with the launch of our new Immunoassay Selection Guide tool. Search by keyword or filter by assay type, detection method, sample type, protein family, and more.

 

Protocols From Invitrogen Protocols from Invitrogen

Check out the Invitrogen™ Protocols web area today. Here you will find detailed protocols for Invitrogen™, Molecular Probes®, Gibco®, and Dynal® products, and much more. Protocols for a broad range of research areas and applications are being added every day. Don’t see what you need? Request a protocol and provide feedback.



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