A superior dye for live cell cycle analysis

what it is
Vybrant® DyeCycle™ Ruby Stain is a near-infrared emission stain for DNA content analysis, enabling live cell cycle analysis with limited cytotoxicity.

how it works
Vybrant® DyeCycle™ Ruby Stain is a dye that penetrates the cell membrane of intact cells. Once inside the cells, the dye binds covalently to the DNA in the nucleus. Staining is simple—suspended cells are incubated with Vybrant® DyeCycle™ Ruby Stain and fluorescence is measured directly. No additional treatment or centrifugation is required. This dye takes advantage of the commonly available 488 nm and 633/635 nm excitation sources with emission >670 nm, leaving common blue laser channels open for other studies. And unlike DRAQ5, Vybrant® DyeCycle™ Ruby Stain does not kill the analyzed cells, offering the possibility of cell sorting based on DNA content.

what it offers
  • live cell cycle analysis
  • limited cell toxicity offers cell sorting potential
  • compatible with any flow cytometer

Excitation and emission peaks of DyeCycle Ruby

Excitation and emission spectra of Vybrant® DyeCycle™ Ruby Stain.

Product Quantity Cat. no.
Vybrant® DyeCycle™ Ruby Stain 400 assays V10273
Vybrant® DyeCycle™ Ruby Stain 100 assays V10309

Faster, more accurate dead cell discrimination

what it is
The SYTOX® AADvanced™ Dead Cell Stain Kit includes a new high-affinity nucleic acid stain that selectively penetrates cells with compromised plasma membranes. The stain can also be used with fixed cells for DNA content cell cycle analysis.

how it works
SYTOX® AADvanced™ Dead Cell stain is spectrally similar to 7-AAD, but with rapid uptake kinetics. After brief incubation with SYTOX® AADvanced™ stain, the nucleic acids of dead cells fluoresce bright red-orange when excited with 488 nm blue laser light. These properties, combined with its >500-fold fluorescence enhancement upon nucleic acid binding, make the SYTOX® AADvanced™ stain a simple and quantitative single-step dead-cell indicator. The DNA content histogram of fixed cells gives lower CVs than 7-AAD, making it ideal for use in multicolor applications requiring DNA content measurement.

what it offers
  • fast labeling of dead cells—only 5 minutes to results
  • efficient cell penetration—better separation of live and dead cells than 7-AAD
  • compatibility with multicolor applications—minimal compensation with the PE channel
  • tighter CVs for more accurate DNA content measurement

SYTOX® AADvanced™ Dead Cell Stain

Discrimination of dead cells using the SYTOX® AADvanced™ Dead Cell Stain Kit. A mixture of heat-killed and untreated Jurkat cells were stained with 1 µM of SYTOX® AADvanced™ Dead Cell stain solution for 5 min. Cells were analyzed on a flow cytometer equipped with a 488 nm laser and a 695/40 nm bandpass filter. Live cells are easily distinguished from the dead cell population.

SYTOX® AADvanced™ Stain

DNA content analysis using the SYTOX® AADvanced™ Dead Cell Stain Kit. HL-60 human promyeloblastic leukemia cells were alcohol fixed and then suspended in 0.1% Triton X/PBS/1% BSA. Cells were stained with 1 µM of SYTOX® AADvanced™ Dead Cell stain solution with the addition of RNase A for 30 min at room temperature. Cells were analyzed on a flow cytometer equipped with a 488 nm laser and a 695/40 nm bandpass filter.

Product Quantity Cat. no.
SYTOX® AADvanced™ Dead Cell Stain Kit
100 assays S10349
SYTOX® AADvanced™ Dead Cell Stain Kit
500 assays S10274

Accurate cell identification with HCS NuclearMask™ Deep Red stain

what it is
The versatile HCS NuclearMask™ Deep Red nuclear stain enables analysis of DNA content and cell demarcation of live and formaldehyde-fixed cells, using high-content imaging and analysis platforms. 

how it works
In image-based high-content screening (HCS) assays, cell or object identification is the first step of the automated image acquisition and analysis process. For many software algorithms, the cell identification process begins with the detection of fluorescent stained nuclei. Using the position of the stained nucleus as a guide, the software then extrapolates to mark the probable position of the cytoplasmic region. With its near-infrared excitation/emission of 638/686 nm when bound to DNA, HCS NuclearMask™ Deep Red stain is ideal for cell demarcation and DNA content analysis, and can be easily discriminated from blue and green fluorophores in multiparametric studies.

what it offers
  • proven performance—validated for image segmentation on HCS platforms
  • selection—alternative to blue-fluorescent Hoechst for multiplexing flexibility
  • simple, user-friendly protocol

HCS NuclearMask™ Deep Red stain

Multiparametric analysis of nocodazole-induced mitotic arrest in A549 cells. Nuclear segmentation and DNA content measurements were performed using HCS NuclearMask™ Deep Red stain. The strong increase in both phospho-H3 and DNA is indicative of mitotic cells. The images were quantitated using the Cellomics® ArrayScan® VTI platform (Thermo Scientific).

Product Quantity Cat. no.
HCS NuclearMask™ Deep Red stain 400 µl H10294

Cell health and toxicity in high-content imaging and analysis

what it is
The HCS LIVE/DEAD® Green Kit provides an ideal means to measure cell viability for high-content imaging studies.

how it works
This kit includes Image-iT® DEAD Green™ viability stain for discrimination of dead cells and HCS NuclearMask™ Deep Red stain or Hoechst 33342 for total cell demarcation. Image-iT® DEAD Green™ viability stain is impermeant to healthy cells, but gains entrance when the plasma membrane integrity of cells is compromised by drugs, test compounds, or other agents. Unlike other viability stains, the Image-iT® DEAD Green™ viability stain is amenable to fixation and permeabilization.

what it offers
  • comprehensive analysis of cell health and toxicity— simultaneous detection of prelethal and lethal aspects of cellular stress and viability
  • content-rich results—includes reagents compatible with fixation and detergent-based permeabilization, enabling the addition of other antibody-based parameters to the assay
  • simple, user-friendly protocol

HCS LIVE/DEAD® Green   Dose response for valinomycin in HeLa cells using the HCS LIVE/DEAD® Green Kit. HeLa cells were treated with valinomycin at final concentrations between 0 to 120 μM and incubated for 24 hr at 37°C. Imaging and analysis were performed using a 10x objective and the Compartmental Analysis BioApplication with the Thermo Scientific Cellomics® ArrayScan® VTI platform.

Product Quantity Cat. no.
HCS LIVE/DEAD® Green Kit 1 kit H10290

Antibodies for cell signaling, neurobiology, and oncology research

what they are
This month we are launching a diverse array of new antibodies covering a broad spectrum of research areas, including nuclear receptors/transcription factors, organelles/cell structure, protein translation/ubiquitination, ion channels, neurobiology/neurodegeneration, kinase/phosphatase proteins, and cell cycle/apoptosis. 

how they work

We are actively expanding our collection of primary antibodies to enable analysis of key targets in normal and disease states, including cardiovascular disease, cancer, inflammation, neurodegenerative diseases, and diabetes. Sixteen new antibodies are available for targets including Stargazin, Aquaporin 2, NMDA, Parkin, and various members of the GABA family. Many of these markers play important roles in signaling pathways leading toward neurodegeneration.

what they offer
  • confidence—all phosphorylation site specificities have been verified with peptide competition
  • performance—robust antibodies are validated for multiple applications
  • selection—a large menu of unique specificities, with a wide range of targets and modification sites

Human SH-SY5Y cells
  Human SH-SY5Y cells stained with mouse anti-fibrillarin, showing prominent blue nucleolar staining. The nuclei are stained with blue-fluorescent DAPI. Cells are also stained with a fluorescently labeled chicken antibody to neurofilament NF-H (red fluorescence).

Western blot of HeLa cell lysate
  Western blot of a HeLa cell lysate showing specific immunolabeling of the ~34 kDa fibrillarin protein.

Product Quantity Applications* Species
Cat. no.
Mouse anti-IL-8 (guinea pig) 100 μg ELISA, WB Hu, GP 433310
Mouse anti-TNF-α (guinea pig) 500 μg ELISA GP 433230
Fibrillarin (Nop1p), Mouse Monoclonal Antibody 100 μl WB, IF/ICC Hu, R, Dr 480009
Tubulin, beta III, Mouse Monoclonal Antibody 100 μl WB, IHC, (FFPE) R, B, Hu, Ms 480011
Ubiquitin C-terminal Hydrolase 1 (UCHL1), Mouse Monoclonal Antibody 100 μl WB, IF/ICC R 480012
Stargazin, Rabbit Polyclonal Antibody 100 μl WB R 480013
GABAA , α3-Subunit, Rabbit Polyclonal Antibody 100 μl WB R, Hu 480016
GABA Transporter (GAT) 2, Rabbit Polyclonal Antibody 100 μl IHC R 480017
GABA Transporter (GAT) 3, Rabbit Polyclonal Antibody 100 μl WB, IHC R 480018
Aquaporin 2 [pS261], Phospho Antibody 100 μl WB R 480019
GABAB [pS783], R2-Subunit, Phospho Antibody 100 μl WB, IF/ICC R 480020
GABAB  [pS923], R1-Subunit, Phospho Antibody 100 μl WB R 480021
NMDA Receptor NR2B Subunit [pS1480], Phospho Antibody 100 μl WB R 480022
MEK5 [pSpT311/315], Phospho Antibody 100 μl WB R 480024
PAK-1,2,3 [pT402], Phospho Antibody 100 μl WB R 480025
Parkin [pS101], Phospho Antibody 100 μl WB Hu 480026
Parkin [pS378], Phospho Antibody 100 μl WB Hu 480027
Polo-Like Kinase 1 [pS210], Phospho Antibody 100 μl WB Hu, R 480028
NMDA NR2A, N-terminus, Rabbit Polyclonal Antibody 100 μl WB R 480031
* ICC = immunocytochemistry, IF = immunofluorescence, IHC = immunohistochemistry, WB = western blot.
† B = bovine, Dr = Drosophila, GP = guinea pig, Hu = human, Ms = mouse, R = rat.

For a list of additional phosphorylation site–specific antibodies, visit www.invitrogen.com/pssa.
To browse antibodies by specificity or application, visit www.invitrogen.com/antibodies.
Click a spotlight onto hidden posttranslational modifications

what it is
The Click-iT® Protein Reaction Buffer Kit provides researchers with proteins labeled with a terminal azide or alkyne and the corresponding click detection reagent with the power to “click” these two moieties together. Each kit includes sufficient materials to perform 25 reactions for subsequent analysis of the protein by gel electrophoresis, western blot, or mass spectrometry.

how it works
Click chemistry describes a powerful new class of chemical reactions that use biologically unique moieties to label and detect proteins of interest using a simple, two-step procedure. In the first step, an azide- or alkyne-containing biomolecule is fed to cells and then actively incorporated into the protein. Unlike other labels, the azide and alkyne tags are small enough that tagged biomolecules (e.g., sugars and amino acids) are acceptable substrates for the enzymes that incorporate these building blocks into proteins. A subsequent detection step uses the chemoselective ligation or “click” reaction between an azide and alkyne, where the modified protein is detected with a corresponding azide- or alkyne-containing dye or hapten for subsequent analysis by standard biochemistry techniques such as gel electrophoresis or western blotting.

what it offers
  • pioneering biological insights—expose previously undiscovered and elusive posttranslational modifications
  • exquisite sensitivity—detect femtomole protein levels
  • context-rich results—perform multiplexed analyses in the same gel or blot

Overview of the Click azide/alkyne reaction
Overview of the click chemistry azide/alkyne reaction. The azide and alkyne moieties are interchangeable, allowing the molecule to be labeled with an alkyne and then react with a fluorophore- or hapten-azide.

Product Quantity Cat. no.
Click-iT® Protein Reaction Buffer Kit 1 kit C10276

The AbC™ Anti-Mouse Bead Kit for accurate flow cytometry compensation

what it is

The AbC™ Anti-Mouse Bead Kit provides a consistent, accurate, and easy-to-use technique
for setting flow cytometry compensation when using fluorochrome-conjugated mouse
antibodies. The kit contains two types of specially modified polystyrene microspheres: the
AbC™ capture beads, which bind all isotypes of mouse immunoglobulin, and negative beads with no antibody-binding capacity.
how it works
After incubation with the same fluorochrome-conjugated mouse antibody that will be used for cell staining, the beads are washed in staining buffer, a drop containing negative control beads is added if required, and the beads are resuspended and analyzed by flow cytometry. The two components provide distinct positive and negative populations of beads that can be used to set compensation.

what it offers
  • fast and simple bead-based compensation
  • elimination of the need to use precious samples for setting compensation
  • accurate and consistent results—highest reactivity to different subclasses of mouse immunoglobulin
  • confidence in your results—avoid inconsistencies due to variations in antigen expression

Compensation using the AbC™ anti-Mouse Bead Kit
Compensation using the AbC™ Anti-Mouse Bead Kit. (A) AbC™ capture beads were labeled with phycoerythrin-conjugated mouse anti-human CD56 antibody ( MHCD56044) for a positive signal, and AbC™ negative beads provided a negative signal. (B) AbC™ capture beads were labeled with FITC-conjugated mouse anti-human CD3 antibody (MHCD03014) for a positive signal, and AbC™ negative beads provided a negative signal. (C) Dual parameter plot showing gated human lymphocytes labeled with phycoerythrin-conjugated mouse anti-human CD56 and FITC-conjugated mouse anti-human CD3 using compensation settings obtained with the AbC™ Anti-Mouse Bead Kit.

Product Quantity Cat. no.
AbC™ Anti-Mouse Bead Kit 1 kit A10344

Anti–green fluorescent protein antibodies

Expression of the intrinsically fluorescent green-fluorescent protein (GFP) from the jellyfish Aequorea victoria has become a highly trusted method for following gene expression and protein localization. Invitrogen continues to offer a comprehensive selection of anti-GFP antibodies. The workhorse of our offering is our rabbit polyclonal antibody raised against GFP purified directly from A. victoria. This anti-GFP antibody, available as a complete serum or as an IgG fraction, facilitates the detection of native GFP, recombinant GFP, and GFP-fusion proteins by immunofluorescence, western blot analysis, and immunoprecipitation. Direct conjugates made from the IgG fraction, using our best Alexa Fluor® dyes, are also available. Additional options for your research include two mouse monoclonal antibodies and a chicken IgY fraction.

HeLa cell transfected with pShooter pCMV/myc/mito/GFP
  HeLa cell transfected with pShooter pCMV/myc/mito/GFP, then fixed and permeabilized. Green-fluorescent protein (GFP) localized in the mitochondria was labeled with mouse IgG2a anti-GFP antibody (A11120) and detected with orange-fluorescent Alexa Fluor® 555 goat anti–mouse IgG antibody (A21422), which colocalized with the dim GFP fluorescence. F-actin was labeled with green-fluorescent Alexa Fluor® 488 phalloidin (A12379), and the nucleus was stained with blue-fluorescent DAPI (D1306, D3571, D21490). The sample was mounted using ProLong® Gold antifade reagent (P36930). Some GFP fluorescence is retained in the mitochondria after fixation (top), but immunolabeling and detection greatly improve visualization (bottom).

Product Quantity Cat. no.
Anti-GFP, rabbit serum (polyclonal) 100 μl A6455
Anti-GFP, rabbit IgG fraction 100 μl A11122
Anti-GFP, rabbit IgG fraction, biotin-XX conjugate 100 μl A10259
Anti-GFP, rabbit IgG fraction, HRP conjugate 100 μl A10260
Anti-GFP, rabbit IgG fraction, Alexa Fluor® 488 conjugate 100 μl A21311
Anti-GFP, rabbit IgG fraction, Alexa Fluor® 594 conjugate 100 μl A21312
Anti-GFP, rabbit IgG fraction, Alexa Fluor® 555 conjugate 100 μl A31851
Anti-GFP, rabbit IgG fraction, Alexa Fluor® 647 conjugate 100 μl A31852
Anti-GFP, mouse IgG2a, monoclonal 3E6 100 μg A11120
Anti-GFP, mouse IgG1, monoclonal 11E5 100 μg A11121
Anti-GFP, chicken IgY fraction 100 μl A10262
Anti-GFP, chicken IgY fraction, biotin-XX conjugate 100 μl A10263

Lytic granule loading of CD8+ T cells is required for HIV-infected cell elimination associated with immune control.
Migueles, S.A. et al. (2008) Immunity 29:1009–1021.

What is the biochemical basis of natural HIV resistance? Long-term nonprogressors (LTNPs, or elite controllers) are rare individuals who manifest a natural control over HIV replication. LTNPs have demonstrated the ability to maintain stable CD4+ T cell counts and low viral loads over the course of many years following their infection with HIV, even in the absence of antiretroviral drug therapy. While several studies have suggested a role for HIV-specific CD8+ T cells in the successful control of HIV replication in LTNPs, details of their role remain poorly understood.  In the present study, Migueles and colleagues investigated the cytotoxicity of HIV-specific CD8+ T cells from LTNPs; they observed that these cells exhibit much greater cytotoxic capacity than those from progressors (i.e., non-resistant individuals). Cytotoxicity assays using the LIVE/DEAD® Fixable Violet Stain Kit linked this enhanced cytotoxic capacity to the granzyme B content of lytic granules. Further experiments showed that the lytic power of progressor CD8+ T cells could be re-established by treating them with phorbol ester and calcium ionophore, an effect possibly mediated by an NFAT (nuclear factor of activated T cells)-related mechanism. The authors suggest that future therapeutic regimens for HIV could involve agents that increase the cytotoxic capacity of HIV-specific CD8+ T cells.

Human breast cancer cells were subjected to a wound-healing assay after cytokine deprivation. The morphology of the cytoskeleton and nucleolar subdomains during cell migration was monitored by an indirect immunofluorescence assay. The cytoskeleton was detected with a rabbit anti-α-tubulin antibody and labeled using Alexa Fluor® 488 goat anti–rabbit IgG (green fluorescence, Cat. no. A11008), nucleolar subdomains were detected with mouse anti-fibrillarin antibody and labeled using Alexa Fluor® 594 goat anti–mouse IgG (red fluorescence, Cat. no. A11005), and nuclei were stained with DAPI (blue fluorescence, Cat. no. D1306). The sample was mounted using ProLong® AntiFade Reagent (Cat. no. P7481) and visualized using epifluorescence microscopy at 400x magnification with a cooled CCD. The low background noise makes it possible to see detailed information about the special arrangements of both the cytoskeleton and nucleoli under a large field of view using a simple epi-ilumination microscope. The photostability of the Alexa Fluor® dye–labeled conjugates mounted in ProLong® AntiFade Reagent allowed ample time for focusing and image capture. Image contributed by Grace L.Y. Chen, School of Medical Laboratory Science and Biotechnology, Taipei Medical University.
High Content Screening - HCS   Find the tools you need for high-content screening
High-content screening (HCS) is a marriage between instrumentation, reagents, and informatics that brings fluorescent microscopy into the higher-throughput domain. Since this new approach was introduced in the late 1990s, Invitrogen Molecular Probes® products have been instrumental for the growth of this field. We recently introduced a portfolio of validated HCS kits for toxicology and cytotoxicity screening. These kits add to our already extensive line of tools and kits for HCS platforms including our LipidTOX™ reagents and CellMask™ stains.

You can find out more about all our HCS reagents at our newly updated HCS central page at www.invitrogen.com/hcs.

MAPK Signaling Pathway   New MAPK signaling pathway web page
Whether your MAPK pathway research requires basic research tools, cell-based assays, or comprehensive screening services, Invitrogen has solutions for you. Empower your research today using our comprehensive portfolio of products and services to investigate the MAPK pathway—everything from high-quality reagents for basic research and assay development to validated biochemical and cell-based assays, as well as world-class profiling and screening services.

View our portfolio of reagents for MAPK signaling at www.invitrogen.com/mapk.

High Content Screening - HCS Free online technical webinars
You are invited to join us for a series of biweekly technical webinars from the comfort of your desk. The webinars will initially focus on imaging-related applications, but we welcome your feedback for additional topics throughout the course of the year. Upcoming topics will be announced each month via email.

Presentations will last approximately 45 minutes, followed by 15 minutes for live Q&A.
Webinars Date Time
New Approaches to Live-Cell Imaging March 3, 2009 10:00 a.m. PST
Microscope Performance and Calibration March 17, 2009 10:00 a.m. PST
Accessorize Your Imaging—Encore Presentation March 31, 2009 10:00 a.m. PST

Missed our February webinars? Find them at the links below:

Molecular Probes® The Handbook

©2009 Molecular Probes, Inc. Visit our website for a complete listing of legal notices and trademark attributions. Feel free to distribute ProbesOnline to friends and colleagues, but please keep this copyright statement intact.