Read the latest issue of the ProbesOnline E-Newsletter.
In This Issue
|No-Wash Apoptosis Imaging — CellEvent™ Caspase-3/7 Green Detection Reagent|
|Sensitive Oxidative Stress Detection — CellROX™ Deep Red Reagent|
|New Antibodies for Secretory Pathway Research — Recombinant Syntaxin (Pep12) Antibodies|
|Multiplex Analysis of Autophagy and Other Cell Functions|
|Zenon® IgG Labeling Kits|
FEATURED NEW PRODUCTS
what it is
The CellEvent™ Caspase-3/7 Green Detection Reagent is a new reagent for imaging apoptosis that does not require any wash steps, is compatible with high-content imaging, and is well suited for multiplex imaging in live or fixed cells.
what it offers
- Simple, no-wash protocol preserves delicate apoptotic cells
- Suitable for live-cell imaging, enabling continuous monitoring of dynamic events
- Compatible with formaldehyde-based fixation methods
how it works
The CellEvent™ Caspase-3/7 Green Detection Reagent consists of the DEVD peptide sequence conjugated to a nucleic acid–binding dye. The substrate is intrinsically nonfluorescent, as the DEVD peptide inhibits the ability of the dye to bind to DNA. In the presence of activated caspase-3/7, the DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright fluorescence response. The fluorescence emission of the dye when bound to DNA is ~530 nm and can be observed using a standard FITC filter set.
- Learn More About the CellEvent™ Caspase-3/7 Green Detection Reagent
Multiplex measurements of apoptosis and oxidative stress.
HepG2 cells were treated with 50 µM nefazodone or DMSO (control) for 24 hr. During the last 30 min of treatment, the cells were stained with 5 µM CellEvent™ Caspase-3/7 Green Detection Reagent, 5 µM CellROX™ Deep Red Reagent, and Hoechst 33342. Treated cells (B) showed clear increases in both oxidative stress (red) and apoptosis (green), compared to control cells (A).
|CellEvent™ Caspase-3/7 Green Detection Reagent, 2 mM solution in DMSO||100 µL||C10423|
CellROX™ Deep Red Reagent is a novel fluorogenic probe for detecting oxidative stress in cells. The bright, deep red–fluorescent signal is compatible with other live-cell dyes and GFP, making it useful in multiplex fluorescence assays to measure a variety of cellular phenomena, including cytotoxicity and cell death. Unlike many other ROS sensors, the signal from CellROX™ Deep Red Reagent is retained after formaldehyde fixation.
what it offers
Compatible with GFP and Alexa Fluor® 488 dye
- Suitable for live-cell imaging or formaldehyde-based fixation
- Validated with multiple platforms
The cell-permeant CellROX™ dye is nonfluorescent in a reduced state and exhibits bright fluorescence upon oxidation by reactive oxygen species. Its emission maximum of ~665 nm is measurable by fluorescence imaging, high-content imaging, fluorescence plate readers, or flow cytometry.
- Learn More About CellROX™ Deep Red Reagent
Detection of oxidative stress using CellROX™ Deep Red Reagent.
BPAE cells were plated in a 96-well plate, treated with 100 μM menadione for 1 hr to induce oxidative stress, then stained with 5 μM CellROX ™ Deep Red Reagent, 20 nM MitoTracker® Green, and Hoechst 33342 for 30 min in complete medium. Cells were then washed 3 times with PBS. The control cells (left) show no signal, while the menadione-treated cells (right) show a robust increase in signal as a result of oxidative stress.
|CellROX™ Deep Red Reagent, for oxidative stress detection||5 x 50 µL||C10422|
what they are
Yeast syntaxin (Pep12) is a key component of the endocytic pathway. Localized in the prevacuolar compartment, syntaxin plays a role in trafficking endocytic cellular contents between early or recycling endosomes to the vacuole. Dysfunction in vesicle trafficking has been recently implicated in metabolic diseases. We now offer highly specific ABfinity™ recombinant antibodies against syntaxin.
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. This 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
Western blot detection of yeast syntaxin.
Syntaxin was detected with Syntaxin PEP12 Abfinity™ Recombinant Rabbit Monoclonal Antibody (left) or Syntaxin PEP12 Abfinity™ Recombinant Rabbit Oligoclonal Antibody (right). Antibodies were used at 1 µg/mL with 1 ng/lane Rec Yeast Syntaxin+Fusion Protein. The western detection was performed using a WesternBreeze® kit with NBT/BCIP as the substrate.
|Syntaxin PEP12 ABfinity™ Recombinant Rabbit Monoclonal Antibody||100 µg||700689|
|Syntaxin PEP12 Recombinant Rabbit Oligoclonal Antibody||100 µg||710037|
A full understanding of autophagy requires the measurement of changes in other cellular events that occur in parallel. The LC3B Antibody Kit for Autophagy, when used with Click-iT® kits, allows researchers to study a myriad of cellular events—including cell proliferation, RNA transcription, protein synthesis, protein lifetime, apoptosis, and posttranslational modifications of proteins—in conjunction with autophagy.
The LC3B protein normally resides in the cytosol. During autophagy, LC3B is cleaved and lipidated with phosphatidylethanolamine, and associates with the autophagosome as it forms. This association with the autophagosome makes it one of the most reliable markers for autophagy. Click-iT® kits can be used to measure cell proliferation (Click-iT® EdU), nascent protein synthesis (Click-iT® AHA), apoptosis (Click-iT® TUNEL), changes in newly synthesized RNA (Click-iT® EU), and posttranslationally modified proteins (via the incorporation of various "clickable" sugar analogs). Click-iT® detection occurs through the copper-catalyzed covalent reaction between an azide and an alkyne, one of which bears a fluorescent reporter such as an Alexa Fluor® dye. Click-iT® kits are available in a variety of colors and are multiplexable with the LC3B Antibody Kit as well as many other assays, markers, and antibodies.
||Multiplex analysis of LC3B accumulation and other parameters of cell function.
The induction and inhibition of autophagy can be combined with other measures of cell health. Click labeling was combined with LC3B staining in HeLa cells after treatment with chloroquine. Click-iT® detection was used to label (A) proliferating cells (Click-iT® EdU, pink; rabbit anti-LC3B, green), (B) protein lifetime (Click-iT® AHA, green; rabbit anti-LC3B, red), (C) RNA transcription (Click-iT® RNA, pink; rabbit anti-LC3B, purple), or (D) posttranslational modification with N-acetylated galactosylamine (Click-iT® GalNAz, green; rabbit anti-LC3B, blue). EdU and EU were fed to cells after drug treatment, whereas GalNAz and AHA were fed to cells before drug treatment. All Click-iT® reactions were carried out using Alexa Fluor® 488 dye conjugated to either an alkyne or azide. Cells were costained with Hoechst 33342, anti-LC3B rabbit IgG, and Alexa Fluor® 647 dye–conjugated goat anti-rabbit secondary antibodies.
|LC3B Antibody Kit for Autophagy||1 kit||L10382|
|Click-iT® EdU Alexa Fluor® 488 Imaging Kit||1 kit||C10337|
|Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay, 2-plate size||1 kit||C10289|
|Click-iT® RNA Alexa Fluor® 488 HCS Assay, 2-plate size||1 kit||C10327|
|Click-iT® EdU Alexa Fluor® 488 HCS Assay, 2-plate size||1 kit||C10350|
|Click-iT® GalNAz Metabolic Glycoprotein Labeling Reagent (tetraacetylated N-azidoacetylgalactosamine), for O-linked glycoproteins||5.2 mg||C33365|
|Zenon® IgG Labeling Kits are the fastest way to noncovalently label very small amounts of rabbit and mouse IgG antibodies with fluorophores, biotin, or fluorescent proteins (e.g., R-PE). Zenon® labeling technology uses a labeled Fab fragment directed against the Fc portion of an intact IgG antibody to form a labeling complex. Because the Zenon® labeling method is based on immunoselectivity, there is no need to remove exogenous proteins or amine-containing buffers from the antibody sample before forming the complex.
The entire labeling procedure takes only 10 minutes, and nearly 100% of the primary antibody is labeled. Zenon® labeling technology dramatically simplifies time-consuming immunocytochemical applications such as the use of multiple same-species–derived primary antibodies in one staining experiment. The extensive selection of available labels and the versatility of Zenon® technology make it easy to experiment with different color combinations for optimal multicolor images. Quick and convenient, Zenon® labeling requires only 1 µg of primary antibody for the entire labeling procedure. The resulting Zenon® complex is also fully compatible with most flow cytometry applications.
Use of Zenon® labeling technology in fluorescence imaging. Fixed and permeabilized bovine pulmonary artery endothelial cells were stained with Alexa Fluor® 594 phalloidin and Hoechst 33342 to label actin filaments and the nucleus, respectively. Anti–OxPhos Complex V inhibitor protein antibody was complexed with the Zenon® Alexa Fluor® 488 Mouse IgG1 Labeling Kit to stain the mitochondria.
|Fluorescence SpectraViewer — There’s an App for That
Plot and compare spectra, check spectral compatibility, and email the configuration to yourself or others in the lab. This mobile SpectraViewer acts as an extension of our online Fluorescence SpectraViewer:
||Imaging the Golgi complex, mitochondria, and cytoskeleton in fibroblast cells. Albino Swiss mouse embryo fibroblast cells (3T3) were stained with an Alexa Fluor® 750–conjugated anti-giantin antibody (Golgi, yellow; prepared using Alexa Fluor® 750 succinimidyl ester), MitoTracker® Deep Red stain (mitochondria, blue), Alexa Fluor® 488 Phalloidin (actin, green), an Alexa Fluor® 569–conjugated anti-vimentin antibody (intermediate filaments, red), and Hoechst 33258 (nucleus, cyan). Image contributed my Michael W. Davidson, Florida State University.|
|Alexa Fluor® 488 Phalloidin||300 units||A12379|
|Hoechst 33258, 10 mg/mL solution in water||10 mL||H3569|
|MitoTracker® Deep Red FM, special packaging||20 x 50 µg||M22426|
|Alexa Fluor® 750, succinimidyl ester||1 mg||A20011|
Amplex® UltraRed Enhances the Sensitivity of Fluorimetric Pyruvate Detection
Zhu A, Romero R, Petty HR (2010) Anal Biochem 403:123–125.
Indicators of intracellular pyruvate levels are important tools for investigating cell growth, signaling, and metabolism. One assay commonly used to measure pyruvate includes horseradish peroxidase (HRP) and pyruvate oxidase, which have better activity in acidic environments than in basic or neutral solutions. Zhu and colleagues recently reported the results of pyruvate measurements obtained using the fluorescent indicator Amplex® UltraRed Reagent. Because the Amplex® UltraRed reaction product has a lower pKa than resorufin (the product of the previous indicator, Amplex® Red), the researchers were able to collect data using lower-pH buffers, which are more suited to the enzymes in their assays. The authors also noted that the Amplex® UltraRed reaction product exhibited higher fluorescence over the pH 5–7 range compared to resorufin, and higher Z′-factor values (0.93 for Amplex® UltraRed compared to 0.89 for Amplex® Red).
|Amplex® UltraRed Reagent||5 x 1 mg||A36006|
The Molecular Probes® Handbook
FOR RESEARCH USE ONLY. NOT INTENDED FOR ANY ANIMAL OR HUMAN THERAPEUTIC OR DIAGNOSTIC USE.
© 2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Feel free to distribute ProbesOnline to friends and colleagues, but please keep this copyright statement intact.