ProbesOnline™ April 2014

In this issue


Premo™ Cellular Hydrogen Peroxide Sensor   Dynamic measurement of hydrogen peroxide in live cells—Premo™ Cellular Hydrogen Peroxide Sensor
Click-iT® Plus EdU proliferation kits   Improved multiplex compatibility—New Click-iT® Plus EdU proliferation assay kits for flow cytometry
Click-iT® Plus Alexa Fluor® Picolyl Azide Toolkits   Customizable biomolecule detection—Click-iT® Plus Alexa Fluor® Picolyl Azide Toolkits
Alexa Fluor® carboxylic acids   Bright and photostable Alexa Fluor® reference standards—Nonreactive form of Alexa Fluor® carboxylic acids
ReadyProbes® Cell Viability Imaging Kits   Convenient, ready-to-use kits for cell viability—ReadyProbes® Cell Viability Imaging Kits




BioProbes® Journal of Cell Biology Applications

The Molecular Probes® Handbook


Wisconsin Stem Cell Symposium
April 30, 2014
BioPharmaceutical Technology Center
Madison, WI, USA

American Association of Immunologists (AAI)
May 2–6, 2014
David L. Lawrence Convention Center
Pittsburgh, PA, USA

International Society for Advancement of Cytometry (ISAC/CYTO)
May 17–21, 2014
Greater Fort Lauderdale/Broward County Convention Center
Fort Lauderdale, FL, USA


Dynamic measurement of hydrogen peroxide in live cells

Premo™ Cellular Hydrogen Peroxide Sensor

What it is
The new Premo™ Cellular Hydrogen Peroxide Sensor is a fluorescent protein–based reporter that detects hydrogen peroxide (H2O2) in living cells in a dynamic and reversible manner. The Premo™ H2O2 sensor combines the selectivity of roGFP-Orp1 chimeras for H2O2 with the transduction efficiency of BacMam 2.0 technology for ease of use with different cell lines. The sensor can be readily combined with other probes for powerful multiplex assays in cell biology.

What it offers

  • High selectivity for hydrogen peroxide
  • BacMam 2.0 gene delivery technology allows easy and efficient expression in most cell types
  • Dynamic and reversible measurement of hydrogen peroxide in live cells
  • Improved accuracy using ratiometric detection

How it works
roGFP is a modified GFP with two cysteines introduced into its β-barrel structure, rendering this protein sensitive to oxidation and useful in probing reactive oxygen species (ROS). Upon oxidation, these cysteines form a disulfide bridge leading to protonation of the roGFP and a shift in the excitation maximum from 488 nm (of normal GFP) to 400 nm. This shift in the excitation maximum permits ratiometric detection of the fluorescence emission at 515 nm. Orp1 is an H2O2-sensitive yeast protein that forms disulfides upon highly selective reaction with H2O2, and this protein has the ability to transfer signals by disulfide exchange to other redox-sensitive proteins [1] such as  roGFP.  Orp1, expressed as a fusion with roGFP, serves as a “molecular antenna” to convey H2O2 selectivity to roGFP in live cells.

  1. Gutscher et al. (2009) J Biol Chem 284:31532–31540.
Premo™ Cellular Hydrogen Peroxide Sensor  
Time and dose response curves for Premo™ Cellular Hydrogen Peroxide Sensor.
U2OS cells were transduced with Premo™ Cellular Hydrogen Peroxide Sensor; after 48 hours, time-lapse imaging was done on a Zeiss® LSM 710 confocal microscope. Images were captured every 15 seconds, over a 10-minute time span after addition of varying doses of H2O2, using alternating 400 nm and 488 nm excitation. Fluorescence emission was captured at 515 nm. Fluorescence intensity values were quantified by marking regions of interest on the cells and were used to calculate 400 nm/488 nm excitation ratios. The ratios were plotted against time.

Improved multiplex compatibility

New Click-iT® Plus EdU proliferation assay kits for flow cytometry

What they are
The Click-iT® Plus EdU flow cytometry kits provide a simplified and more robust assay for analyzing DNA replication in proliferating cells compared to traditional BrdU methods. Unlike the original Click-iT® EdU assay and some BrdU assays, the Click-iT® Plus EdU assay can be used in conjunction with R-PE and R-PE tandems, as well as fluorescent proteins such as GFP and mCherry.

What they offer

  • Multiplexable—unlike BrdU or the original Click-iT® EdU, Click-iT® Plus EdU is completely compatible with R-PE, R-PE tandems, and fluorescent proteins
  • Accurate—superior results compared to BrdU assays
  • Fast—results in as little as 60 minutes

How they work
The Click-iT® Plus EdU proliferation assay is a novel alternative to the BrdU assay. EdU (5-ethynyl-2′-deoxyuridine) is a thymidine analog that is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry, which is a copper-catalyzed covalent reaction between an azide and an alkyne. The Click-iT® Plus reaction uses a modified azide in place of the azide used in the original Click-iT® reaction. As a result of the modification, the concentration of free copper in the sample is significantly lower and fluorescence signals from fluorescent proteins (e.g., R-PE, R-PE tandem dyes, and GFP) are not quenched. The speed and accuracy of the Click-iT® Plus EdU reaction is comparable to that of the original Click-iT® EdU reaction.

  • Learn more about Click-iT® Plus EdU assay kits for flow cytometry
Click-iT® Plus EdU Alexa Fluor® 488 Kit Compatibility of Click-iT® Plus EdU reaction with R-PE tandem conjugate fluorescence. Jurkat (human T-cell leukemia) cells were treated with EdU for 2 hours and then stained with a PE-Cy®7 conjugate of mouse anti–human CD3 antibody. Newly synthesized DNA was detected according to the protocol included in the Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit. The cells were analyzed on the Attune® Acoustic Focusing Cytometer using 488 nm excitation. The Alexa Fluor® 488 fluorescence signal was detected using a 530/30 nm bandpass emission filter, while the PE-Cy®7 fluorescence signal was detected using a >640 nm longpass filter. The data demonstrate that the PE-Cy®7 fluorescence signal was retained following detection of the incorporated thymidine analog, EdU, with the Click-iT® Plus Alexa Fluor® 488 picolyl azide.

Customizable biomolecule detection

Click-iT® Plus Alexa Fluor® Picolyl Azide Toolkits

What they are
Click-iT® Plus Alexa Fluor® Picolyl Azide Toolkits contain everything you need to perform copper-catalyzed click reactions with compounds that are sensitive to copper, while retaining all of the benefits of the standard azide/alkyne click reaction. Unlike the original Click-iT® azides, picolyl azide–based click reactions minimize the free copper in the click reaction, thus protecting proteins (e.g., GFP, RPE), nucleic acids (e.g., RNA, oligos), and even small molecules (e.g., phalloidin) against undesirable copper side reactions.

What they offer

  • Flexibility—optimize your own Click–iT® reaction for the detection or labeling of any alkyne-containing biomolecule in vitro or in cells and tissue samples
  • Brightness and sensitivity—superior fluorescent Alexa Fluor® dyes combined with the efficiency of picolyl azide
  • Convenience—kits contain all the buffers and reagents needed to create the optimal click reaction

How they work
The Alexa Fluor® picolyl azide incorporates a copper-chelating group to concentrate copper at the reaction site. The copper protectant limits the concentration of free copper ions, accelerates the cycloaddition reaction, and acts as a nontoxic reducing agent to limit further reduction of copper, thus limiting the generation of reactive oxygen species (ROS). By adjusting the ratio of copper and copper protectant, one can control the amount of free copper in the click reaction and optimize it for the sample.

Biomolecule sensitivity to copper
Sensitivity of biomolecules to copper.
The effects of low, medium, and high free copper concentrations on various molecules' function and structure are shown. Green bars represent copper concentrations where the molecule functions normally; yellow and red bars represent the copper concentrations where the molecule function and/or structure is damaged or completely inhibited by copper. With the Click-iT® Plus toolkit, one can adjust the ratio of copper and copper protectant and control the amount of free copper in the click reaction, thereby optimizing it for the sample.

Bright and photostable Alexa Fluor® reference standards

Nonreactive form of Alexa Fluor® carboxylic acids

What it is
Alexa Fluor® dyes are bright, photostable, and water-soluble dyes that can be used for a broad range of applications, including fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy, and spectrofluorometry. We now offer nonreactive Alexa Fluor® carboxylic acids that can be used as reference standards or for directly labeling molecules.

What they offer

  • Brightness and photostability—compared to traditional dyes, Alexa Fluor® dyes are brighter and more photostable
  • pH resistance—fluorescence of the dyes is unchanged between pH 4 and 10
  • Efficiency—the Alexa Fluor® structures allow a higher degree of labeling without intramolecular quenching

How they work

Our Alexa Fluor® carboxylic acids are nonreactive forms of the Alexa Fluor® dyes that can be used as reference standards for dye conjugates. Additionally, these carboxylic acid derivatives can be converted to amine-reactive esters using standard chemical techniques, or be coupled to hydrazines, hydroxylamines, and amines in aqueous solution using water-soluble carbodiimides such as EDAC.

Alexa Fluor® carboxylic acid
Alexa Fluor® carboxylic acid for use as a reference standard.

Convenient, ready-to-use kits for cell viability

ReadyProbes® Cell Viability Imaging Kits

What they are
ReadyProbes® Cell Viability Imaging Kits are ready-to-use reagents designed to help you quickly and easily detect live versus dead cells in your sample. Just add 2 drops of the reagent to 1 mL of your cell sample in growth medium, incubate for 5–20 minutes, and count the number of total cells versus the number of dead cells. ReadyProbes® reagents are formulated for maximum convenience without sacrificing any of their optical or physicochemical properties. These reagents are provided with concise protocols and user-friendly packaging that includes recyclable, recycled, and sustainable materials where possible.

What they offer

  • Exceptionally bright fluorescence when bound to DNA
  • Rapid staining of total and dead cells without wash steps, using a convenient dropper bottle
  • Room temperature–stable formulation eliminates need to dilute, weigh, or pipet

How they work
Each ReadyProbes® Cell Viability Imaging Kit contains a cell-permeant nuclear stain that can cross the plasma membrane of all cells, regardless of whether they are alive or dead. Both of the kits use a blue-fluorescent nuclear stain for the total cell stain. Each kit also contains a cell-impermeant nuclear stain that can only cross the plasma membrane once it has been compromised. See the table below for a summary of the staining pattern seen with each kit.

ReadyProbes® Cell Viability Imaging Kit All cells Dead cells Filters needed
Blue/Red Blue Red DAPI/TRITC
Blue/Green Blue Green DAPI/FITC
ReadyProbes® Cell Viability Imaging Kit  
Detection of cell viability using the ReadyProbes® Cell Viability Imaging Kit. Live (blue fluorescence) and heat-killed (green fluorescence) Jurkat cells were mixed 1:1 to create a sample with 50% live and 50% dead cells. Cells were stained with the ReadyProbes® Cell Viability Imaging Kit (Blue/Green) and incubated for 20 minutes at room temperature. Imaging was performed on the EVOS® FL Auto Imaging System.


Improve phosphoprotein detection using the Attune® Acoustic Focusing Cytometer

The study of mitogen-activated protein kinase (MAPK) is important for investigating diseases such as cancer. MAPK signaling cascades play important roles in critical decision processes within the cell, including cellular responses to environmental stimuli and disease progression.

Multiparameter flow cytometry is a valuable tool for dissecting signaling pathways in cell populations using intracellular staining with fluorescent antibodies against phosphorylation site–specific proteins. While there are reagents and techniques available for phosphoprotein-specific detection, the signals that result from these are usually dim and difficult to distinguish. Advancements in instrumentation using acoustic focusing—the Attune® Acoustic Focusing Cytometer—enable better detection of dim signals than by conventional hydrodynamic focusing. The Attune® cytometer employs high-frequency sound waves to maintain a tightly focused sample stream, allowing greater precision at the laser interrogation point. Using the High Sensitive transit time setting to slow the sample stream allows longer laser interrogation time, which increases the sensitivity of detection.

We used three phosphoproteins, Akt, Erk1/2, and p38, to demonstrate an ideal research application for the Attune® cytometer, which enables the detection of dim signals through highly sensitive and precise data-gathering capabilities.

Attune® Acoustic Focusing Cytometer
Comparison of High Sensitive and Standard transit times (using 25 μL/min sample injection rate) on the Attune® Acoustic Focusing Cytometer to the Low Flow Rate (12 μL/min) of the BD™ LSR II and BD FACSCalibur™ instruments, using Jurkat cells treated with anisomycin and stained with p38 Alexa Fluor® 488 direct conjugate. Purple traces represent untreated, p38 Alexa Fluor® 488–stained Jurkat cells; blue traces represent anisomycin-treated, p38 Alexa Fluor® 488–stained Jurkat cells. The Attune® Acoustic Focusing Cytometer demonstrates improved separation of low-expressed proteins using the High Sensitive mode, compared to the instruments using conventional hydrodynamic focusing. (A) High Sensitive, 25 μL/min, showing unstained, untreated Jurkat cells for reference (red trace), SI = 3.1. (B) High Sensitive, 25 μL/min (unstained cells not shown), SI = 3.1. (C) Standard, 25 μL/min (unstained cells not shown), SI = 2.7. (D) BD FACSCalibur™ 12 μL/min (Low Flow Rate), (unstained cells not shown), SI = 3.1. (E) BD™ LSR II 12 μL/min (Low Flow Rate), (unstained cells not shown), SI = 3.2.


Multiple choices for multiple applications

Anti-GFP antibodies

Expression of the intrinsically fluorescent green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a well-established method for visualizing specific intracellular proteins or cellular structures (e.g., mitochondria, nuclei, cytoskeleton) as well as for monitoring gene expression and other physiological processes (see references). In some instances, the fluorescent signal from GFP can be compromised (low expression) or destroyed (exposure to fixatives or paraffin embedding). In such cases, the GFP molecule may still be intact and can be detected with antibodies against the protein.

With over 400 citations, our unconjugated anti-GFP polyclonal antibodies have been the standard of a proven performer. Our anti-GFP antibodies are generated via isolation of highly purified full-length, native A. victoria protein. After inoculation of GFP into the rabbit host, the rabbit serum is purified and tested for reactivity against both native and denatured forms of GFP.

The GFP Rabbit Serum Polyclonal Antibody, which contains a highly specific antibody against GFP, is provided in complete rabbit serum. For applications requiring optimal performance, the IgG fraction is purified from the serum by ion exchange chromatography to remove nonspecific immunoglobulins. The resulting product, the GFP Rabbit IgG Polyclonal Antibody Fraction, contains only the IgG fraction from the rabbit serum raised against GFP.

The unconjugated anti-GFP polyclonal antibodies have been validated for use in the following applications:

  • Immunohistochemistry (IHC)
  • Immunoprecipitation (IPP)
  • Western analysis
  • Immunocytochemistry (ICC)

Learn more about:

GFP detection in transduced cells
Detection of GFP in transduced cells.
GFP Rabbit IgG Polyclonal Antibody Fraction was labeled with Alexa Fluor® 488 dye. U2OS cells were transduced with CellLight® Mitochondria-GFP, BacMam 2.0. After an overnight incubation the cells were fixed in formaldehyde, permeabilized, and incubated with Alexa Fluor® 488–conjugated GFP antibody (green fluorescence; mitochondria). Cells were then stained with Texas Red®-X phalloidin (red fluorescence; cytoskeleton) and Hoechst 33342 (blue fluorescence; nuclei).


On the web

The Life of Cells  

“Life of Cells” minifigure collectible characters

Introducing Stem Cell, the first of 5 collectible characters from the "Life of Cells” video series to be immortalized in plastic cubes. The "Life of Cells” characters are so engaging, they’re taking on a life of their own. Check out their new online space where you can see videos, play fun games, and more. Find out how you can be one of the first researchers to get your hands on the Stem Cell minifigure.

Meet Stem Cell

Imaging corner

Confocal imaging of human dermal fibroblasts

Confocal imaging of human dermal fibroblasts  
Gibco® Human Dermal Fibroblasts, neonatal (HDFn) were transduced with CellLight® Golgi-GFP, BacMam 2.0 (cyan) and CellLight® Peroxisomes-RFP, BacMam 2.0 (purple) and imaged on a Zeiss® laser scanning confocal microscope with a 63x oil immersion lens.

Highlight from BioProbes® Journal

Evaluate neural stem cells for neurodegenerative disease models: Neural stem cell–specific protein expression and mitochondrial assessment

In the Cell Health section of BioProbes® 70, you will find the article “Evaluate neural stem cells for neurodegenerative disease models”, which discusses two of several analyses performed on neural stem cells (NSCs) during the development of cell models targeted for Parkinson’s disease (PD) research. Our research collaboration with The Parkinson’s Institute (Sunnyvale, California) has set about to develop PD model systems using NSCs derived from induced pluripotent stem cells (iPSCs) that were generated from diseased fibroblasts collected at the institute. The development of suitable PD models would not only accelerate the discovery of disease mechanisms and drug targets but also serve a critical role in screening for clinical and therapeutic strategies.

In this article, we describe two assays for evaluating NSC identity and health. To confirm cell type, we used the Human Neural Stem Cell Immunocytochemistry Kit, which provides reagents and a protocol for convenient image-based analysis of four common markers of human NSCs (nestin, Sox1, Sox2, and Pax6). To assess mitochondrial activity, we treated the NSCs with three different cell stressors in the presence of the red-fluorescent MitoSOX® Red Mitochondrial Superoxide Indicator. See the results of these analyses in the full BioProbes® article, where you can also follow the links to two white papers that show more of the data produced through our collaboration with The Parkinson’s Institute.

Detection of oxidative stress in PD-3 NSCs using MitoSOX® Red Mitochondrial Superoxide Indicator  
Detection of oxidative stress in PD-3 NSCs using MitoSOX® Red Mitochondrial Superoxide Indicator.
PD-3 NSCs were cultured in StemPro® NSC SFM for 24 hr at 37°C in 384-well assay plates coated with CTS™ CELLstart™ substrate. After a 1 hr incubation with 5 μM MitoSOX® Red Mitochondrial Superoxide Indicator, the cells were washed once with growth medium and treated with the stressor compounds for 2 hr; the resultant fluorescence was measured on a Tecan® Safire2™ Fluorescence Plate Reader. The PD-3 NSCs showed the expected increase in oxidative stress with increased concentrations of rotenone, valinomycin, and tert-butyl hydroperoxide (TBHP).

† What's new with the BioProbes® Journal?

We are bringing our award-winning BioProbes® articles to you sooner. We will be publishing new BioProbes® articles online every month and highlighting those articles here. That way, we can keep you up-to-date on new fluorescence technologies and cell biology applications. Check back frequently and watch BioProbes® 70 Journal take shape!



Basics of multicolor flow cytometry panel design

With the proliferation of new fluorescent dyes as well as instruments that can detect 18 or more parameters, multicolor flow cytometry has become more popular and more accessible than ever. This webinar will discuss aspects of good panel design, including:

  • Rules for designing panels
  • Examples and practical application of these rules
  • Controls and standardization
  • Relevance of panel design to new mass cytometry platforms

Presenter: Holden T. Maecker, PhD, Director of the Human Immune Monitoring Center at Stanford University