In This Issue
FEATURED NEW PRODUCTS
Quickly Profile Markers in the Akt Pathway—New Magnetic Akt 7-Plex Panels Easy Detection of Secreted Alkaline Phosphatase—Phospha-Light™ EXP SEAP Reporter Gene Assay A Fix for Your Immunofluorescence Samples—Image-iT® Fixation/Permeabilization Kit New Antibody for Studying Cell Cycle—Ki-67 ABfinity™ Recombinant Rabbit Oligoclonal Antibody
- Select flow reagents by application area
- Visualize predicted results for each reagent
- View streamlined, intuitive protocols
- Track protocol progress with built-in timer
On the Web—Benchtop Devices—Simple Solutions to Everyday Complexities
Imaging Corner—Cytoskeletal and Mitochondrial Staining in Neonatal Epidermal Keratinocytes
From the Bench—Finding the Connections Between Oxidative Stress and DNA Replication
Molecular Probes® Webinar Series—Flow Cytometry Educational Webinars
Association of Biomolecular Resource Facilities (ABRF)
American Association for Cancer Research (AACR)
March 31–April 4
|BioProbes® Journal of Cell Biology Applications|
|The Molecular Probes® Handbook|
FEATURED NEW PRODUCTS
what they are
New Akt Pathway Phospho and Total 7-Plex Magnetic Panels provide accurate quantitation of 7 proteins in cell lysates and tissue homogenates. These panels are suitable for use with the Luminex® 200™, FLEXMAP® 3D, and MAGPIX® systems.
what they offer
- Sample and time savings―measure 7 targets simultaneously in one well
- Superior performance―accurate, reproducible, and sensitive quantitation of multiple proteins
- Precision―high reproducibility, <10% CV for consistency
how they work
Samples are mixed with magnetic beads of defined spectral properties conjugated to analyte-specific capture antibodies, and incubated for 2 hours. After binding, a magnet can be used for easy washing similar to ELISA washing. Analyte-specific biotinylated detector antibodies are then added and incubated with the beads for 1 hour. During this second incubation, the analyte-specific biotinylated detector antibodies recognize the epitopes and bind to the appropriate immobilized analytes. After removal of excess biotinylated detector antibodies, streptavidin conjugated to the fluorescent protein R-phycoerythrin (RPE) is incubated for 30 minutes. After washing, the beads are analyzed using a Luminex® instrument to quantify all 7 analytes.
- Learn more about new Akt Pathway Phospho and Total 7-Plex Magnetic Panels
Evaluation of the performance of the Akt Pathway Phospho 7-Plex Magnetic Panel. MCF-7 cells, Insulin Receptor (IR)–transfected CHO-T cells, and Jurkat cells were treated with insulin or lithium chloride (LiCl) to stimulate phosphorylation of Akt pathway analytes.
|Akt Pathway Phospho 7-Plex Magnetic Panel||Akt [pS473], GSK-3β [pS9], IRS-1 [pS312], IGF-1R [pY1135/pY1136], IR [pY1162/pY1163], p70S6K [pT421/pS424], PRAS40 [pT246]||100 tests||LHO0001M|
|Akt Pathway Total 7-Plex Magnetic Panel||Akt, GSK-3β, IGF-1R, IR, IRS-1, p70S6K, PRAS40||100 tests||LHO0002M|
what it is
The Phospha-Light™ EXP SEAP Reporter Gene Assay Kit using NovaBright™ chemiluminescence technology makes detection of SEAP reporter gene activity easier than ever. The simplified procedure eliminates reagent preparation and sample dilution steps.
what it offers
- Sensitivity—outperforms detection by fluorescence or colorimetric methods
- Assay simplicity—two ready-to-use reagents, no sample dilutions
- Improved signal-to-noise ratio and low-end sensitivity
how it works
The kit combines our high-performance NovaBright™ chemiluminescence alkaline phosphatase substrate, CSPD®, and our next-generation Emerald™ enhancer to provide better assay performance over 5 orders of magnitude of enzyme concentration. Our proprietary buffer formulation minimizes background due to endogenous phosphatases, enabling the Phospha-Light™ EXP SEAP kit to provide the most sensitive detection, with a higher signal-to-noise ratio for SEAP reporter gene activity than other assays of its kind.
- Learn more about the Phospha-Light™ EXP SEAP Reporter Gene Assay Kit
Phospha-Light™ EXP SEAP assay sensitivity and dynamic range compared to other chemiluminescent SEAP reporter gene kits. pCMV-SEAP–transfected NIH 3T3 cells were assayed with Phospha-Light™ EXP (blue), Phospha-Light™ SEAP (pink), supplier A (green), or supplier B (orange) assay kits. The Phospha-Light™ EXP and Phospha-Light™ SEAP kits enable detection of secreted placental alkaline phosphatase with lower limits of detection and better signal-to-noise ratios than other commercially available kits.
|NovaBright™ Phospha-Light™ EXP Assay Kit for SEAP||192 assays
|Phospha-Light™ SEAP Reporter Gene Assay System||400 assays
what it is
The Image-iT® Fixation/Permeabilization Kit contains all of the necessary reagents to prepare your cells for antibody staining and imaging. The high-quality components help preserve cell morphology and reduce background staining, and are provided in a convenient storage box in single-use vials with easy-to-follow protocols.
what it offers
- A complete fixation/permeabilization kit with ready-to-use components and room temperature storage
- Optimized for secondary antibody labeling, fixed-cell dye staining, and applications involving Green Fluorescent Protein (GFP)
- Compatible with analysis of most cellular antigens
how it works
Cells are treated sequentially with the fixative, the permeabilization solution, and the blocking buffer according to a simplified and easy-to-follow protocol. The reagents are optimized to preserve 3D cellular structure and provide adequate antibody access to intracellular antigenic sites. The high-quality components and optimized formulations help decrease background staining, improving signal-to-noise levels and image quality. In extensive testing with both nuclear and cytoplasmic targets, the reagents provided in this kit offered results equal or superior to those from standard reagents.
- Learn more about the Image-iT® Fixation/Permeabilization Kit
Fluorescently labeled cells fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit. (A) Neonatal human dermal fibroblasts were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit. Peroxisomes were detected with an anti-PMP70 antibody and labeled with Alexa Fluor® 488 goat anti–rabbit IgG. Actin was labeled with Alexa Fluor® 594 phalloidin, and the nucleus was stained with Hoechst 33342. Images were acquired using a Nikon® upright microscope (60x). (B) Bovine pulmonary artery endothelial (BPAE) cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit. Mitochondria were detected with ATP Synthase Subunit IF1 Monoclonal Antibody and labeled with Alexa Fluor® 488 goat anti–mouse IgG. Actin was labeled using Alexa Fluor® 594 phalloidin, and the nucleus was stained with Hoechst 33342. Images were acquired on a Nikon® upright microscope (60x).
what it is
Ki-67 is a large (~360 kDa) nonhistone protein that is associated with, and may be required for, cell proliferation. Ki-67 protein is not detected in the G0 phase of the cell cycle but is detected in steadily increasing amounts from the G1 phase through mitosis. Ki-67 accumulates beginning in late G1 and is localized in small granules throughout the nucleus. We now offer Ki-67 ABfinity™ Recombinant Rabbit Oligoclonal Antibody for cell cycle studies.
what it offers
- ABfinity™ recombinant monoclonal and oligoclonal antibodies offer consistent results
- Minimize the need to revalidate working antibody dilutions for your experiments each time you order
how it works
ABfinity™ antibodies are manufactured by transfecting mammalian cells with high-level expression vectors containing immunogen-specific heavy- and light-chain antibody cDNA. This production process offers consistent lot-to-lot antibody performance. ABfinity™ oligoclonal antibodies are a mixture of recombinant monoclonal antibodies, combining the improved signal strength of a polyclonal antibody with the highly reproducible results that ABfinity™ monoclonal antibodies provide.
Immunocytochemical analysis of HeLa cells. Cells were serum starved for 36 hr, followed by serum release for 8 hr. (A) Cells were then labeled with Ki-67 ABfinity™ Recombinant Rabbit Oligoclonal Antibody followed by Alexa Fluor® 488 goat anti–rabbit IgG (green). (B) Nuclei were stained with DAPI (blue). (C) Actin was stained with Alexa Fluor® 594 phalloidin (red). (D) Composite image showing nuclear localization of Ki-67.
|Ki-67 ABfinity™ Recombinant Rabbit Oligoclonal Antibody–Purified||100 µg||710229|
The Tali® Image-Based Cytometer is a valuable tool for routine cell analysis, delivering quantitative data right at your benchtop. In two recent application notes, we demonstrated the versatility of the Tali® Image-Based Cytometer in moving beyond fluorescent protein or viability detection to quantitative analysis of cell cycle and caspase-3/7 activation.
As described in the first application note, quantitative detection of S-phase cells was accomplished using the Click-iT® EdU Alexa Fluor® 555 reagent and the red channel of the Tali® instrument. Mitotic arrest (M phase) was detected using anti-phosphohistone H3 antibody, an Alexa Fluor® 488 secondary antibody, and the green channel of the Tali® instrument. As described in the second application note, apoptotic cells were detected and accurately counted using CellEvent® Caspase-3/7 Green Detection Reagent and the green channel of the Tali® instrument.
CellEvent® Caspase-3/7 Green Detection Reagent analyzed on the Tali® Image-Based Cytometer and the BD™ LSRII flow cytometer. Jurkat cells were treated with 10 µM camptothecin or vehicle control for 5 hr. Analysis by both instruments showed that approximately 52% of camptothecin-treated cells were positive for active caspase-3/7.
The recommended protocols for many fluorescently labeled affinity proteins require diluting the conjugate to a working concentration, and then using the resultant solution immediately. When the diluted solution is not used immediately, performance may become less optimal over a period of hours, depending on the concentration, container material, and buffer formulation. A common laboratory practice for stabilizing diluted solutions is to add minimally interfering carrier proteins. This strategy can also be used to stabilize solutions containing diluted fluorescently labeled proteins.
For example, the fluorescence signals from diluted solutions of fluorescent Qdot® 655, Alexa Fluor® 488, and R-phycoerythrin streptavidin conjugates diminish dramatically over 3 hours in a polystyrene 96-well microtiter plate when diluted in 1X PBS alone. However, adding 1% bovine serum albumin (BSA), 10% fetal bovine serum (FBS), or 10% goat serum (GS) to the buffer dramatically stabilizes the solutions, and fluorescence intensity is maintained for at least hours. Using this technique can help researchers obtain brighter and more consistent fluorescence labeling.
|Enhancing solution stability using protein or serum. Streptavidin conjugates of three fluorophores (Alexa Fluor® 488, Qdot® 655, and R-phycoerythrin) were diluted with 1X PBS (buffer only), 1X PBS containing 1% bovine serum albumin (BSA), 10% fetal bovine serum (FBS), or 10% goat serum (GS). Solutions were transferred to a 96-well polystyrene microtiter plate, and fluorescence intensity was monitored for 3 hr using a TECAN Infinite® M1000 plate reader. The percentage of detectable fluorescence remaining after 3 hr is shown (error bars indicate one standard deviation from the mean). In all cases, diluting with the buffer containing BSA or serum dramatically improved solution stability.|
Benchtop Devices—Simple Solutions to Everyday Complexities
Discover how easy it can be to count cells, quantitate DNA, or even perform PCR. Watch as Luca, a precocious preschooler, demonstrates the use of the Countess® Automated Cell Counter, Qubit® 2.0 Fluorometer, and Veriti® Thermal Cycler.
Cytoskeletal and Mitochondrial Staining in Neonatal Epidermal KeratinocytesHuman neonatal epidermal keratinocytes were grown in EpiLife® medium with Human Keratinocyte Growth Supplement and plated on coverslips. Mitochondria were labeled with MitoTracker® Red CMXRos (yellow). Cells were then fixed with 4% formaldehyde and permeabilized with 0.2% Triton® X-100. Cell junctions were labeled with anti–β-catenin mouse monoclonal antibody and Alexa Fluor® 647 goat anti–mouse IgG (pink), actin was labeled with Alexa Fluor® 488 phalloidin (green), and nuclei were labeled with Hoechst 33342 (blue). Coverslips were mounted using ProLong® Gold Antifade Reagent and imaged on a Zeiss LSM 710 confocal microscope. Image contributed by Kevin Chambers, Life Technologies Corporation.
Finding the Connections Between Oxidative Stress and DNA Replication
Zhao H, Dobrucki J, Rybak P, Traganos F, Halicka HD, Darzynkiewicz Z (2011) Cytometry Part A 79A:897–902.
If not repaired, DNA damage caused by H2O2 and other oxidants accumulates over time and contributes to aging, cellular senescence, and predisposition to disease. In a recent publication, Zhao and colleagues presented results from a study to determine the correlation between damage response events and DNA replication. The researchers pulse-labeled cells with EdU and then exposed them to H2O2. Incorporated EdU was detected using an Alexa Fluor® 488 dye–labeled azide, and multiparameter laser-scanning flow cytometry and confocal microscopy were employed to identify DNA-replicating cells and examine the location of DNA replication sites. Using this approach, the researchers were able to see a close association between DNA replication and damage response events, and postulated that these events may be triggered by stalled replication forks and possibly by induction of DNA double-stranded breaks at the sites of H2O2-induced damage. Detecting incorporated EdU using click chemistry made it possible to identify cells that are replicating their DNA and to examine other intracellular epitopes at the same time.
- View the bibliography reference
- The Click-iT® EdU Alexa Fluor® 488 Imaging Kit and EdU were used in this study for click chemistry labeling
- Alexa Fluor® 568 goat anti–mouse IgG secondary antibody was used for immunocytochemical labeling
Flow Cytometry Educational Webinars
Learn the basics of flow cytometric analysis in two free, recorded webinars.
Basics of Flow Cytometry, Part II
FOR RESEARCH USE ONLY. NOT INTENDED FOR ANY ANIMAL OR HUMAN THERAPEUTIC OR DIAGNOSTIC USE.
© 2012 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.
* Compatible with iPhone® and Android® operating systems. App is provided “AS IS” without any warranty. Installation or use of the app is subject to terms and conditions.