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In This Issue
|Distinguish Dead from Live Cells in Flow Cytometry Assays — SYTOX® Dead Cell Stains|
|Identify Downstream Interferon Signaling Events — ABfinity™ Recombinant Monoclonal Antibodies for JAK-STAT Targets|
|Application Notes for the Countess® Automated Cell Counter
|Become a Molecular Probes® Handbook Facebook Fan!
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Buzzworthy — SYTO® Probes in the Cytometry of Tumor Cell Death
The View — Transverse Section of Fixed Zebrafish Retina
What's New — Attune® Cytometer Named One of Top 10 Innovations in 2010
- On the Web — Immunoassay Selection Guide, Invitrogen™ Protocols
Northwest Flow Cytometry Meeting
Oregon Health & Science University
Expanded Content Now Online
FEATURED NEW PRODUCTS
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.
- Learn More About 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 Maximum||Emission Maximum||Cat. No.|
|SYTOX® Green Dead Cell Stain for flow cytometry||488 nm||504 nm||523 nm||S34860|
|SYTOX® Orange Dead Cell Stain for flow cytometry||488, 532 nm||547 nm||570 nm||S34861|
|SYTOX® Dead Cell Stain Sampler Kit||Various||Various||Various||S34862|
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.
- Learn More About ABfinity™ Recombinant Rabbit Monoclonal Antibodies
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.
|STAT1 [pY701] ABfinity™ Recombinant Rabbit Monoclonal Antibody, Purified||Human
|STAT4 ABfinity™ Recombinant Rabbit Monoclonal Antibody, Purified
|STAT6 [pY641] ABfinity™ Recombinant Rabbit Monoclonal Antibody, Purified||Human||100 µg||700247|
|JAK1 [pY1022/pY1023] ABfinity™ Recombinant Rabbit Monoclonal Antibody, Purified
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 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.
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. 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.
|Low-density lipoprotein from human plasma (LDL), 2.5 mg/mL
|Low-density lipoprotein from human plasma, acetylated, Alexa Fluor® 488 conjugate (Alexa Fluor® 488 AcLDL)
|Low-density lipoprotein from human plasma, acetylated, Alexa Fluor® 594 conjugate
|Transferrin from human serum, Alexa Fluor® 594 conjugate
|Transferrin from human serum, Alexa Fluor® 488 conjugate
|FM® 1-43FX, fixable analog of FM® 1-43 membrane stain
||10 x 100 μg
|DiOC18 (3,3′-dioctadecyloxacarbocyanine perchlorate)
|Dextran, pHrodo™ 10,000 MW for endocytosis
|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:
|Countess® Automated Cell Counter
|Countess® Automated Cell Counter Starter Kit with 11 boxes of slides
|Countess® Automated Cell Counter Starter Kit with 101 boxes of slides
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.
click to enlarge
|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).
|ELF® 97 Immunohistochemistry Kit||1 kit||E6600|
|Hoechst 33342, trihydrochloride, trihydrate||100 mg||H1399|
|Hoechst 33342, trihydrochloride, trihydrate, FluoroPure™ grade||100 mg||H21492|
|Hoechst 33342, trihydrochloride, trihydrate, 10 mg/mL solution in water||10 mL||H3570|
|Wheat germ agglutinin, tetramethylrhodamine conjugate||5 mg||W849|
|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
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