ProbesOnline newsletter

In this issue


  Measure cytosolic pH—pHrodo™ Green AM and pHrodo™ Red AM intracellular pH indicators
  Fluo-4 now in a complete kit for imaging applications—Fluo-4 Calcium Imaging Kit
  The next generation of membrane potential probes has arrived—FluoVolt™ Membrane Potential Kit
  Multiplex western blotting with fluorescent antibody conjugates—Westerns of many colors with WesternDot® secondary antibodies
  Antibody conjugates for studying inflammation by flow cytometry—New mouse monoclonal anti–human CD284 (TLR4) antibody conjugates



  Optimize fluorescence imaging with the best antifade protection—ProLong® Gold and SlowFade® Gold antifade reagents

Measure cytosolic pH

pHrodo™ Green AM and pHrodo™ Red AM intracellular pH indicators

What they are
pHrodo™ Green AM and pHrodo™ Red AM are novel fluorogenic probes used to measure intracellular pH in live cells. pHrodo™ Green and pHrodo™ Red are weakly fluorescent at neutral pH, but their fluorescence increases as the pH drops. These reagents can be used to determine cellular cytosolic pH in the range of pH 4–9. Their photostability and very good retention by cells eliminate the need to perform ratiometric measurements, making the workflow faster and easier than workflows requiring ratiometric techniques.

What they offer

  • Nonratiometric measurement of intracellular pH in live cells
  • Can be multiplexed with other fluorescent indicators
  • Compatible with most fluorescence microscopes and other fluorescence instruments
  • Excellent photostability and retention within cells once the acetoxymethyl (AM) group is removed by intracellular esterases

How they work
The pHrodo™ Green and pHrodo™ Red dyes have been modified with AM ester groups, which results in uncharged molecules that can permeate cell membranes. Once inside the cell, the lipophilic AM ester blocking groups are cleaved by nonspecific esterases, resulting in charged dyes that are retained within the intracellular space. The fluorescence intensity of the probe is then an indicator of intracellular pH. If determination of specific pH is needed, the Intracellular pH Calibration Buffer Kit can be used to calibrate either of these indicators.

pHrodo™ Green AM and pHrodo™ Red AM intracellular pH indicators are compatible with various platforms, including traditional fluorescence microscopy, high-content screening (HCS), flow cytometry, and microplate-based fluorometry or high-throughput screening (HTS). These reagents are also compatible with various Life Technologies™ benchtop instruments such as the EVOS®, FLoid®, Tali®, and Attune® instruments.

pHrodo™ Red dye–labeled particles and pHrodo™ Green dye for imaging pH changes in vesicles and cytosol, respectively. Murine J774A.1 macrophages were labeled with pHrodo™ Red 10,000 MW dextran (visualized in top panels) and pHrodo™ Green AM Intracellular pH Indicator (visualized in bottom panels). The pH was clamped to the indicated values using the Intracellular pH Calibration Buffer Kit.

Fluo-4 now in a complete kit for imaging applications

Fluo-4 Calcium Imaging Kit

What it is
The Fluo-4 Calcium Imaging Kit has been designed for the specific detection of calcium flux in imaging applications. Upon binding of calcium to the fluo-4 dye, a large fluorescence emission increase (>100x) can be detected. To save you time, we have collected all the reagents you will need for the detection of calcium flux in live cells.

What it offers

  • Emission from calcium-bound fluo-4 dye (excitation/emission maxima ~494/506 nm) can be detected using standard fluorescein (FITC) filters.
  • Upon binding calcium, the fluorescence intensity of fluo-4 increases more than 100-fold, with a minimal shift in the wavelength maximum.
  • The kit contains all necessary reagents, thus reducing the time to experimental results.

How it works
The Fluo-4 Calcium Imaging Kit has been formulated, optimized, and contains all the necessary reagents for the detection of calcium flux in imaging applications. The fluo-4 dye, an analog of fluo-3, exhibits a greater than 100-fold increase in fluorescence upon binding calcium. For ease of use, the fluo-4 AM dye is supplied as a 1,000X solution in DMSO. In addition to fluo-4 AM, the kit contains 100X PowerLoad™ Concentrate for easy cell loading, a 10X Neuro Background Suppressor, and probenecid.

Fluo-4 Calcium Imaging Kit   Calcium ion flux visualized. Cytosolic calcium flux from neuronal cells detected using the Fluo-4 Calcium Imaging Kit.  

The next generation of membrane potential probes has arrived

FluoVolt™ Membrane Potential Kit

What it is
The FluoVolt™ membrane potential dye represents the next generation in voltage-sensitive fluorescent probes, displaying the best characteristics of the fast- and slow-response membrane potential fluorescent probes. The FluoVolt™ probe can respond to changes in membrane potential in less than a millisecond and displays a high-magnitude response.

What it offers

  • Fast—sub-millisecond response to changes in membrane potential
  • Highly sensitive—response range is typically 25% per 100 mV
  • Instrument-friendly—the FluoVolt™ probe is excited and emission is measured using standard fluorescein (FITC) settings
  • Versatile—can be used in imaging or patch clamp applications

How it works
The FluoVolt™ membrane potential dye is a fast-response probe with a superior potential-dependent fluorescence response. The response is fast enough to detect transient (millisecond) potential changes in excitable cells, and the probe generates a signal change in excess of 25% per 100 mV. The FluoVolt™ membrane potential dye can be used for imaging electrical activity from intact heart tissues, mapping membrane potentials along neurons and muscle fibers, or measuring potential changes in response to pharmacological stimuli.

Membrane voltage changes measured using the FluoVolt™ Membrane Potential Kit. In panels A and B, differentiated NG-108 cells (mouse neuroblastoma–rat glioma hybrid) were loaded with the FluoVolt™ membrane potential dye. (A) Cells were imaged with 10-millisecond illumination pulses and images acquired with 2x binning. The three selected traces (B) show fluorogenic responses from the dye as the selected cells (numbered dots in A) spontaneously depolarize and repolarize in culture. For the traces in panels C and D, human HEK 293 cells (embryonic kidney) were loaded with the FluoVolt™ membrane potential dye, imaged with 10-millisecond illumination pulses, and data acquired with 2x binning. Traces show fluorogenic responses as cells are depolarized at 2-second intervals from –100 mV to +30 mV (C) or in single steps from –80 mV to 0 mV at 2-second intervals (D) .

Multiplex western blotting with fluorescent antibody conjugates

Westerns of many colors with WesternDot® secondary antibodies

What they are
WesternDot® fluorescently labeled secondary antibodies can be detected on a broad range of gel and blot imaging platforms. They are designed to deliver superior signal-to-noise ratios and exhibit sensitivity comparable to enhanced chemiluminescence (ECL)-based methods in western blot applications. No stripping and reprobing or multiple blots are required for multiple protein detection.

What they offer

  • Versatility—up to 3-color multiplexing
  • Reduced time-to-results—eliminate cumbersome HRP and ECL optimization, and use any fluorescent gel or blot imager you have available
  • Easy to switch—WesternDot® antibodies are compatible with standard membranes, blocking solutions, and buffers and can be detected with a fluorescent gel or blot imager

How they work
Differently labeled WesternDot® antibodies can be applied to a single blot for simultaneous detection of multiple proteins using standard gel or blot imaging platforms. Four colors in the red to near-IR allow detection beyond autofluorescence of western blot membranes, resulting in low background and superior signal to noise. Our WesternDot® antibodies use Qdot® nanocrystals enhanced with VIVID® technology, making them brighter than the original Qdot® reagents. They can be detected with any equipment that has blue or UV light excitation and filters appropriate for ethidium bromide or SYBR® Safe dye.

  Simultaneous detection of EGFR and phospho-EGFR on a single blot using WesternDot® secondary antibody conjugates. A western blot containing triplicate samples (20 μg protein) of lysates from unstimulated (lanes 1–3) and hEGF-stimulated (lanes 4–6) A431 cells was probed with mouse anti-EGFR and rabbit anti–phospho-EGFR antibodies, followed by WesternDot® 800 goat anti–mouse IgG (pseudocolored green) and WesternDot® 655 goat anti–rabbit IgG (pseudocolored red) conjugates. The merged image shows overlaid red and green bands as yellow bands. The blot was imaged using the Fujifilm® LAS-4000 gel imager.

Antibody conjugates for studying inflammation by flow cytometry

New mouse monoclonal anti–human CD284 (TLR4) antibody conjugates

What they are
The Molecular Probes® portfolio of over 1,700 highly specific primary antibodies for flow cytometry is expanding to include more Research Use Only (RUO) selections. The mouse anti–human CD284 (TLR4) antibodies are now available as conjugates to expand your options for studying innate immunity and inflammation.

What they offer

  • Trust—the Molecular Probes® brand
  • Validation—all antibodies are tested in flow cytometry applications
  • Selection—expanded offerings of primary antibody conjugates for flow cytometry

CD284 (TLR4) is a member of the Toll-like receptor family of type I transmembrane proteins, characterized by an extracellular domain with leucine-rich repeats and a cytoplasmic domain with homology to the type I IL-1 receptor. TLR4 is a pattern recognition receptor and signaling molecule that responds to bacterial lipoproteins, suggesting it may play a role in innate immunity and inflammation. TLR4 forms a complex with MD-2 and CD14 for LPS recognition and signaling. TLR4 is expressed at low levels on the surface of peripheral blood monocytes and even lower levels on other cell types, including granulocytes and immature dendritic cells (iDC). However, a relatively high degree of donor variability has been reported for TLR4 expression.



Staining of cells transfected with human TLR4. Transfected cells were probed with Mouse IgG2a kappa PE-Cy®7 conjugate isotype control (open histogram) or anti–human CD284 (TLR4), PE-Cy®7 conjugate (filled histogram). Total viable cells were used for analysis.


Growth of flagellar filaments of Escherichia coli is independent of filament length

Turner L, Stern AS, Berg HC (2012) J Bacteriol 194(10):2437–2442.

A bacterial flagellum consists of three parts: a basal body that is embedded in the cell wall, a short “hook” just outside the cell wall, and a long filament composed of flagellin proteins in a long helical structure. Flagellar filaments grow from the distal end, with the flagellin proteins being synthesized in the bacterium and fed through the central channel of the helix, by a proton-motive force, to self-assemble at the flagellum tip. Based on limited electron microscopy [1] and dark-field light microscopy [2] evidence, and because the flagellin building blocks have to traverse the length of the flagellum’s helical core, flagellar growth rate has long been thought to slow exponentially as the filament length increases.

However, Turner et al. [3] have recently developed an elegant method to measure flagellar filament growth rates using fluorescent probes, and used the method to demonstrate that the growth rates are independent of filament length. They start with bacteria that have an engineered flagellin gene that substitutes a cysteine for a serine in the resulting flagellin protein. This permits the flagella to be labeled with thiol-reactive fluorescent dyes. In the image below, they labeled exponentially growing bacteria with green-fluorescent maleimide (thiol-reactive) dye, then followed with an exposure to red-fluorescent maleimide dye for a defined period of growth. Note that the lengths of the red segments of flagella are all about the same, regardless of the lengths of the green segments. The rate of growth of the flagellar helix is not dependent on the length of the filament.

  1. Iino T (1974) Assembly of Salmonella flagellin in vitro and in vivo. J Supramol Struct 2:372–384.
  2. Aizawa S-I, Kubori T (1998) Bacterial flagellation and cell division. Genes Cells 3:625–634.
  3. Turner L, Stern AS, Berg HC (2012) Growth of flagellar filaments of Escherichia coli is independent of filament length. J Bacteriol 194(10):2437–2442.
  Growth of Escherichia coli flagella imaged by fluorescence microscopy. Flagellar filaments of E. coli strain HCB1737 were labeled with thiol-reactive Alexa Fluor® 488 C5 maleimide (green), cultured for an additional period of time, and then labeled with Alexa Fluor® 546 C5 maleimide (red). Image provided by Howard C. Berg, Alan S. Stern, and Lynda Turner, Harvard University, Cambridge, Massachusetts, and reproduced with permission. A similar figure and more information can be found in Turner et al. (2012), J Bacteriol 194(10):2437–2442.


Optimize fluorescence imaging with the best antifade protection

ProLong® Gold and SlowFade® Gold Antifade Reagents

ProLong® Gold and SlowFade® Gold Antifade Reagents suppress photobleaching and preserve the signals of your fluorescently labeled target molecules. For immediate viewing choose SlowFade® Gold, but for the best fluorescence imaging and for long-term storage of fluorescent samples on slides, choose ProLong® Gold.

ProLong® Gold and SlowFade® Gold reagents are designed to provide maximum photobleaching protection across the whole spectrum typically used for fluorescence imaging. Compared with conventional DABCO formulations or competitor products such as Vectashield® mounting medium, ProLong® Gold and SlowFade® Gold Antifade Reagents show superior maintenance of initial signal strength—particularly of red fluorophores—when tested side by side with a panel of fluorescent antibody conjugates on glass slides (see figure). Also, ProLong® Gold and SlowFade® Gold Antifade Reagents do not produce autofluorescence and are compatible with most of the fluorescent proteins, including GFP, RFP, YFP, and CFP.

  • Ready-to-use formulations are stable at room temperature and provided in dropper bottles
  • Available with or without DAPI for easy nuclear counterstaining
  • For convenience, now also available in 1 x 2 mL format

Learn more about mounting media and antifade reagents

SlowFade® Gold antifade reagent   Comparison of signal retention after mounting using three antifade formulations. A panel of equimolar concentrations of dyes coupled to IgG was evenly arrayed using a non-contacting spot array device (Biochip Arrayer BCA1 from PerkinElmer). The arrayed spots were then overlaid with three test mountants (SlowFade® Gold antifade reagent, Vectashield® mounting medium, and a DABCO formulation) and coverslipped. Fluorescence intensities were measured before and after mounting. SlowFade® Gold Antifade Reagent maintains initial intensity levels over the broadest range of dyes. In contrast, Vectashield® mounting medium and the DABCO formulation can quench >90% of the intensity of some red-fluorescent dyes.


On the web

Newly revised mobile apps—Quickly find what you need for flow cytometry or imaging applications

The newly revised Molecular Probes® Flow Cytometry and Molecular Probes® Fluorescence Imaging mobile apps are designed to help you find fluorescent reagents, kits, and protocols for cell biology–related applications. Now you can use your mobile device to:

  • Select reagents by application area or product type
  • See predictive results for each reagent or kit
  • View streamlined, intuitive protocols
  • Track protocol progress with a built-in timer that runs in the background, even if you leave the app

The mobile apps also include fluorescence excitation and emission data for each product, helping to make experimental design and setup easier than ever. Listed below are the application areas and product types included in both apps.

Application areas Product types
  • Cell viability
  • Cell proliferation
  • Cell cycle
  • Cell tracing and tracking
  • Apoptosis
  • Oxidative stress
  • Phagocytosis
  • Protein labeling kits
  • Primary antibodies
  • Secondary antibodies
  • Calibration tools
  • Instruments

Download mobile apps:

Molecular Probes® Flow Cytometry Reagent Guide and Protocols Molecular Probes® Fluorescence Imaging Reagent Guide and Protocols

Learn more about:

Imaging corner

Four-color fluorescence cell staining highlighting mitochondria, peroxisomes, actin, and nuclei


MitoTracker® Orange CMTMRos was prepared at 300 nM and incubated for 30 min with live BPAE cells in complete medium to label mitochondria (orange). Cells were then fixed and permeabilized, labeled with a primary rabbit polyclonal to PMP70, and visualized with Alexa Fluor® 647 goat anti–rabbit IgG (H+L) antibody (pink). Cells were also stained with ActinGreen™ 488 ReadyProbes® Reagent (green) and NucBlue® Fixed Cell Stain (blue). The image was acquired at 40x magnification on a Nikon® E800 upright microscope.

Highlight from BioProbes® Journal

Checking vital signs: Molecular Probes® cell health assays for flow cytometry

In the Flow Cytometry section of BioProbes® 69, you will find the article “Checking vital signs: Don’t let dead cells mislead you”, which summarizes Molecular Probes® cell health assays for flow cytometry. Here we describe how the inadvertent inclusion of sick or dead cells in experiments can dramatically affect the outcomes. For example, including dead cells in immunophenotyping experiments can distort the analysis, especially for rare phenotypes. Life Technologies has developed an extensive array of Molecular Probes® stains and kits that allow you to assess critical parameters of cell health and viability using a flow cytometry platform:

  • Use membrane integrity as a discriminator of live and dead cells with SYTOX® Dead Cell Stains and LIVE/DEAD® Fixable Dead Cell Stains.
  • Analyze cell cycle using FxCycle™ and Vybrant® DyeCycle™ dyes.
  • Measure nucleoside incorporation with the Click-iT® EdU Flow Cytometry Assays.
  • Monitor cell proliferation via generational analysis with CellTrace™ dyes.
  • Track apoptosis using the CellEvent® Caspase-3/7 Green assay.

Whether the health of cells is your primary question or simply a critical factor in obtaining accurate results, Molecular Probes® dyes offer a variety of ways to gauge cell viability, cell vitality, and other aspects of cell health.


Exclusion of dead cells eliminates staining artifacts from analysis. After the application of a lymphocyte gate (A), live and dead cells were discriminated using LIVE/DEAD® Fixable Violet Dead Cell Stain Kit (B). Note the significant number of dead cells despite a scatter gate. Subsequent analysis of dead cells (C) and live cells (D) shows the dramatic difference in apparent phenotypes between two cell populations. Reprinted from Perfetto SP, Chattopadhyay PK, Lamoreaux L et al. (2006) J Immunol Methods 313:199–208, with permission from Elsevier.

† 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!



Free on-demand webinar—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 on-demand webinar discusses the requirements 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
View the webinar