ProbesOnline—A Molecular Probes Publication

FLoid™ Cell Imaging Station   Click. Print. Love.—The FLoid® Cell Imaging Station Is That Easy
Attune® Acoustic Focusing Cytometer   Attune® Acoustic Focusing Cytometer—Now Available in Blue/Red Laser Configuration
The New MAGPIX® System   Simple, Cost-Effective Immunoassay Multiplexing—The New MAGPIX® System
Qubit® 2.0 Fluorometer   Sensitive and Accurate Nucleic Acid Quantitation—Introducing the Qubit® 2.0 Fluorometer and the Qubit® Fluorometric Platform
CellROX™ Deep Red Reagent   Sensitive Oxidative Stress Detection—CellROX™ Deep Red Reagent
CellEvent™ Caspase-3/7 Green Detection Reagent   No-Wash Apoptosis Imaging—CellEvent™ Caspase-3/7 Green Detection Reagent
ABfinity™ Monoclonal Antibodies  

New Antibodies for the MAPK Signaling Pathway—ABfinity™ Monoclonal Antibodies

pHrodo™ Red Labeling Kit and pHrodo™ Red Avidin   Fluorogenic pH Sensors for Live-Cell Analyses—pHrodo™ Red Labeling Kit and pHrodo™ Red Avidin
Click-iT® EdU   Proliferation Results Within 90 Minutes—Simplified Click-iT® EdU Kits for Flow Cytometry


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Click. Print. Love.—The FLoid® Cell Imaging Station Is That Easy

What it is
The FLoid® Cell Imaging Station provides easy access to fluorescence imaging. Designed in collaboration with fluorescence microscopy users, the FLoid® device captures high-quality fluorescence images right at your bench. The interface is so simple that even novices can collect three-color overlay images in a few minutes.

What it offers

  • Simplicity—collect and process images using an intuitive user interface, and print data images that peel and stick directly into your notebook
  • Accessibility—capture fluorescent cell images at your bench, not in the darkroom
  • Robustness—protect expensive confocal microscopes from excessive day-to-day use

How it works
The FLoid® Cell Imaging Station features a bright-field relief phase and three fluorescence channels. The innovative design allows imaging using a variety of sample vessels, from microscope slides to multiwell plates to T-flasks. Images from each channel can be processed, overlaid, and printed with a few clicks. Data can be saved as image overlays or as separate images from the individual channels, in several file formats. The interface features seven languages and a reagent guide with protocols, information, and reference images across 20 biological categories.

FLoid Imaging Station  
The FLoid® Cell Imaging Station.

Attune® Acoustic Focusing Cytometer—Now Available in Blue/Red Laser Configuration

What it is
With the Attune® Acoustic Focusing Cytometer, you can get the best of two worlds: high sample-throughput rate, and high sensitivity for analyzing precious samples or detecting rare events. The Attune® cytometer is now available with a blue/red laser configuration, so you can select the configuration that best fits your research needs.

What it offers

  • Precision—high collection rates without compromising CV
  • Speed—rare-event detection up to 10 times faster than other cytometers
  • Simplicity—no-lyse, no-wash method avoids cell loss

How it works
Using the Attune® cytometer with the red laser option, analysis of stained mouse whole blood with a stain/lyse protocol shows excellent separation of cell populations into subsets for immunophenotyping experiments using up to 6 colors. There is strong signal separation for data clarity, and 6-color detection is easily performed with the automated compensation module. In addition, the Attune® cytometer generates less waste than other systems do, due to its low sheath fluid usage per run.

Attune® Acoustic Focusing Cytometer
Six-color immunophenotyping analysis on the Attune® Acoustic Focusing Cytometer with red laser option.
Mouse (C57BL/6J) peripheral blood is stained with a 6-color immunophenotyping panel of anti-mouse direct dye conjugates: CD3, hamster anti-mouse (Alexa Fluor® 647 dye); CD19, rat anti-mouse (PE-Cy®5.5 dye); CD4 PE-Cy®7 dye; and CD8a, rat anti-mouse (Alexa Fluor® 488 dye). Gating was performed on lymphocytes for each of the bivariate plots. The plots show a 2-color representation of the 6-color panel, identifying the major T and B cell subsets of the lymphocyte populations.

Simple, Cost-Effective Immunoassay Multiplexing—The New MAGPIX® System

what it is
The MAGPIX® system is a versatile, affordable, and compact fluorescence detection system based on proven Luminex® xMAP® bead-based technology. The MAGPIX® system is capable of performing up to 50 multiplex immunoassays on a single biological sample. It helps deliver fast, accurate, and reproducible results while saving time and cost and conserving valuable samples.

What it offers

  • Ideal immunoassay tool—simultaneous analysis of up to 50 proteins from a limited volume of precious samples
  • Excellent performance—accurate, reproducible, and sensitive quantification of proteins
  • Reduced time and cost—compared to multiple- and single-analyte ELISAs or western blots
  • Ease of use—designed to work with a broad and expanding range of Invitrogen™ immunoassay magnetic bead kits 

How it works
The MAGPIX® system captures data from singleplex and multiplex immunoassays. It is ideal for both new and experienced Luminex® technology users. The MAGPIX® instrument makes use of novel light-emitting diodes (LEDs) for excitation and CCD camera technology for bead and analyte detection. As a result, the MAGPIX® system is inexpensive and occupies minimal bench space. The system also has streamlined startup and shutdown protocols and requires minimal maintenance, making it easy to operate.

The MAGPIX® System  
The MAGPIX® System.
This compact and affordable Luminex® system performs up 50 assays simultaneously in a single well of a 96-well plate.

Sensitive and Accurate Nucleic Acid Quantitation—Introducing the Qubit® 2.0 Fluorometer and the Qubit® Fluorometric Platform

What it is
The Qubit® Fluorometric Platform combines the newly redeveloped Qubit® 2.0 Fluorometer with the Qubit® assays for nucleic acid quantitation. The instrument and assays have been seamlessly integrated to create a quantitation platform that is far more sensitive and accurate than UV absorbance. Qubit® assays (previously known as Quant-iT™ assays) are compatible with both Qubit® 1.0 and Qubit® 2.0 Fluorometers.

What it offers

  • Larger LCD color touch screen
  • Automated data logging and USB port for efficient data management
  • Display of a standard curve after the calibration
  • Step-by-step workflow navigation 

How it works
As a result of customer feedback, the next-generation Qubit® 2.0 Fluorometer has an improved user interface and data storage and transfer mechanisms, making the instrument more intuitive and easy to use. Qubit® assays, which utilize the proven performance of Molecular Probes® fluorescent dyes, complete the seamless integration of the platform, resulting in a high level of accuracy and specificity for nucleic acid quantitation.

Qubit™ assays   Selectivity of Qubit® assays compared to UV spectrophotometry. Triplicate samples containing lambda DNA (10 ng/μL) and varying amounts of E. coli ribosomal RNA (0–100 ng/μL) were assayed using Qubit® DNA BR and Qubit® RNA BR assays according to kit protocols. The same samples were subsequently measured in triplicate using a NanoDrop® ND-1000 Spectrophotometer, and single measurements were made using a PerkinElmer Lambda 35 Spectrophotometer. The red and orange trendlines indicate the actual concentrations of DNA and RNA, respectively, in the starting samples. The data indicate that UV analysis cannot distinguish between DNA and RNA.

Product Quantity Cat. No.
Qubit® 2.0 Fluorometer 1 instrument Q32866
Qubit® 2.0 Quantitation Starter Kit 1 kit Q32871

Sensitive Oxidative Stress Detection—CellROX™ Deep Red Reagent

What it is
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 sensors of reactive oxygen species (ROS), 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  

How it works
The cell-permeant CellROX™ dye is nonfluorescent in its reduced state and exhibits bright fluorescence upon oxidation by ROS. Its emission at ~665 nm is measurable by fluorescence imaging, high-content imaging, fluorescence plate readers, and flow cytometry.

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. Control cells (left) show no signal, while the menadione-treated cells (right) show an increase in signal as a result of oxidative stress.

No-Wash Apoptosis Imaging—CellEvent™ Caspase-3/7 Green Detection Reagent

What it is
The CellEvent™ Caspase-3/7 Green Detection Reagent is a new reagent for apoptosis imaging 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 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 bright fluorescence. The fluorescence emission of the dye when bound to DNA is ~530 nm and can be observed using a standard FITC filter set.

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).

New Antibodies for the MAPK Signaling Pathway—ABfinity™ Monoclonal Antibodies

What they are
The mitogen-activated protein kinase (MAPK) pathway mediates signal transduction from cell surface receptors to downstream transcription factors, leading to cellular responses such as cell proliferation, growth, motility, survival, and apoptosis. We offer new antibodies for detection of phosphorylation events around key targets within this pathway.

What they offer
ABfinity™ monoclonal antibodies are rigorously validated across multiple applications. The process for manufacturing these antibodies is designed to decrease any lot-to-lot inconsistency in biological reactivity, which results in less variability in your experiments.

How they work
ABfinity™ monoclonal antibodies are recombinant antibodies developed by immunizing animals, screening for functionality, and cloning the immunogen-specific antibody genes into high-level expression vectors. The antibodies are produced on a large scale by expressing them in mammalian cells and purifying them with protein A. Just like antibodies isolated from serum or produced by hybridomas, ABfinity™ recombinant monoclonal antibodies are highly specific for their immunogen. The intact IgG appears on a nonreducing gel as a ~150 kDa band, and upon reduction resolves into a ~25 kDa light-chain band and a ~50 kDa heavy-chain band.

ABfinity™ Monoclonal Antibodies
Immunocytochemistry of pervanadate-treated HeLa cells labeled with rabbit anti-FAK [pY861].
Pervanadate-treated HeLa cells were labeled with rabbit anti-FAK [pY861] (10 µg/mL), which was detected using Alexa Fluor® 488 goat anti–rabbit IgG (green, A) at a dilution of 1:1,000. Actin was stained with Alexa Fluor® 594 phalloidin (red, B), and nuclei were stained with DAPI (blue, C). (D) Composite of panels A, B, and C.

Fluorogenic pH Sensors for Live-Cell Analyses—pHrodo™ Red Labeling Kit and pHrodo™ Red Avidin

What they are
The popular pHrodo™ dye, a fluorogenic pH sensor for endocytosis in live-cell studies, is now offered in two additional formats: in a microscale antibody/protein labeling kit, and as an avidin conjugate for use with any biotinylated target. pHrodo™ dye exhibits pH-sensitive red fluorescence that increases in intensity with increasing acidity. pHrodo™ dye is essentially nonfluorescent in the extracellular environment but exhibits bright red fluorescence at pH 5–7 as it is endocytosed. Although we call the dye pHrodo™ Red, these products contain the same pHrodo™ dye as our pHrodo™ SE; pHrodo™ dextran, and pHrodo™ BioParticles® for phagocytosis.

What they offer

  • Flexibility to create your own probes for investigations of phagocytosis and endocytosis
  • Compatibility for multiplexing with green dyes such as GFP, fluo-4, and calcein
  • Designed and tested for imaging, flow cytometry, and HTS/HCS (microplate) applications

How they work
pHrodo™ Red Avidin and the pHrodo™ Red Microscale Labeling Kit offer rapid means to make any material, including proteins, peptides, viruses, and small molecules, a pHrodo™ Red conjugate. The labeling kit includes reagents for 3 labeling reactions of 20–100 µg protein, with excellent recovery, and can also be used for labeling viruses. Alternatively, pHrodo™ Red Avidin can be used to create bioconjugates using the easy, high-affinity avidin–biotin interaction. Use pHrodo™ Red Avidin when a biotinylated target already exists.

pHrodo™ dye
pHrodo™ dye is ideal for detecting changes in pH.
(A) pHrodo™ dye exhibits pH-sensitive fluorescence. (B) Endocytic vesicles undergo acidification during the maturation process.

Proliferation Results Within 90 Minutes—Simplified Click-iT® EdU Kits for Flow Cytometry

what they are
New streamlined kits using the Click-iT® EdU Flow Cytometry Assay for cell proliferation analysis  are available. Standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® detection reagent to access DNA and label the incorporated EdU, making direct S-phase measurements available typically in less than 90 minutes.

what they offer

  • Streamlined step-by-step protocols
  • Two kit sizes: 50- and 100-test kits
  • Results typically obtained within 90 minutes

how they work
Incorporation of EdU provides a direct measurement of new DNA synthesis in cells, and detection using click chemistry eliminates the need to denature DNA. The Click-iT® advantage is in the chemistry—small, unique, low-background labeling and detection moieties react specifically and covalently, offering consistent results.

Click-iT® EdU Alexa Fluor® 488 and FxCycle™ Violet fluorescence  
Dual-parameter plot of Click-iT® EdU Alexa Fluor® 488 and FxCycle™ Violet fluorescence.

Product Quantity Cat. No.
Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit 50 assays C10425
100 assays C10420
Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit 50 assays C10424
100 assays C10419
Click-iT® EdU Pacific Blue™ Flow Cytometry Assay Kit 50 assays C10418


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