In this issue


BlockAid™ Blocking Solution   A superior blocking solution for immunolabeling cells and tissues—BlockAid™ Blocking Solution
ABfinity™ recombinant antibodies   ABfinity™ recombinant antibodies—New antibody for mTOR serine/threonine kinase
Flow cytometry apoptosis antibodies   Antibody conjugates for studying apoptosis by flow cytometry—New mouse monoclonal anti–human Bcl2 and TRAIL-R1, -R2, -R3, and -R4 conjugates
Recombinant proteins   Over 1,500 recombinant proteins just added—Sino Biological® recombinant proteins brought to you by Life Technologies
Cell Sorting Set-up Beads   New fluorescent microsphere reference beads for your flow cytometer—Cell Sorting Set-up Beads and AlignFlow™ Flow Cytometry Alignment Beads


EVOS® FL Auto Cell counting in color
—EVOS® FL Auto Imaging System



BioProbes® Journal of Cell Biology Applications

The Molecular Probes® Handbook


A superior blocking solution for immunolabeling cells and tissues

BlockAid™ Blocking Solution

What it is
BlockAid™ Blocking Solution is an optimized mix of protein-blocking components for use with cell or tissue immunofluorescence. Minimizing background from nonspecific binding of antibodies is essential for improved sensitivity and maximum signal-to-background ratios. This is especially crucial when looking for low-expressing antigens, when using samples with high autofluorescence, or for techniques where signals are dimmer (such as use of directly labeled primary antibodies or use of super-resolution imaging).

What it offers

  • Excellent background reduction—superior to conventional blocking solutions (see figure)
  • Ready to use—no dilution or stock preparation required
  • Versatile—use with any primary or secondary antibody, streptavidin conjugate, microsphere–protein conjugate, or Qdot® nanocrystal conjugate

How it works
BlockAid™ Blocking Solution is superior to commonly used blocking reagents such as bovine serum albumin, normal serum, or casein, for cells or tissue sections (see figure). No dilution is required, and it can be used in an initial blocking step before the primary antibody is applied, or as a diluent for both the primary and secondary antibody. BlockAid™ Blocking Solution has also been validated for use with streptavidin conjugates, microsphere–protein conjugates, and Qdot® nanocrystal conjugates.

Unwanted fluorescence from nonspecific antibody binding in the presence of various blocking reagents. Mouse cerebellum cryosections were exposed to 5 µg/mL of Alexa Fluor® 488 goat anti–mouse IgG (no other antibody was added) and blocked with 6% bovine serum albumin (BSA), 5% normal goat serum (NGS), or BlockAid™ Blocking Solution, and compared to autofluorescence. Comparable fields were imaged with a 10x objective and the same exposure time. Intensity was quantitated using threshold measurement and plotted (relative fluorescence units).    

ABfinity™ recombinant antibodies

New antibody for mTOR serine/threonine kinase

What they are
ABfinity™ recombinant monoclonal and oligoclonal antibodies offer consistent results, minimizing the need to revalidate working antibody dilutions for your experiments each time you order. Life Technologies currently offers hundreds of ABfinity™ recombinant antibodies, and we are actively developing more.

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a key role in cell growth, cell proliferation, and protein synthesis. mTOR mediates phosphoinositide 3-kinase and Akt/PKB signaling, resulting in phosphorylation of 4EBP1 and initiation of mRNA translation. A second pathway involves regulation of ribosomal S6 kinase, which affects ribosome biogenesis and translation elongation.

What they offer

  • Specificity—undergo rigorous validation
  • High performance—proven consistency from lot to lot
  • Efficiency—detect low-level targets with a small sample

How they work
ABfinity™ antibodies are produced by transfecting mammalian cells with high-level expression vectors containing immunogen-specific rabbit antibody heavy- and light-chain cDNA. This highly reproducible process results in superb consistency in lot-to-lot antibody performance.

ABfinity™ oligoclonal antibodies are mixtures of recombinant monoclonal antibodies. These combine the improved signal strength that can come from using polyclonal antibodies, with the highly reproducible results you get from ABfinity™ monoclonal antibodies.

Intracellular flow cytometry analysis of HeLa cells labeled with ABfinity™ mTOR Recombinant Rabbit Monoclonal Antibody.
Fixed and permeabilized HeLa cells were probed with ABfinity™ mTOR Recombinant Rabbit Monoclonal Antibody, followed by labeling with Alexa Fluor® 488 goat anti–rabbit IgG (right peak, filled purple). To confirm specificity, the cells were probed with an isotype control and labeled using Alexa Fluor® 488 goat anti–rabbit IgG (middle peak, black). Unstained cells form the left peak (blue).

Antibody conjugates for studying apoptosis by flow cytometry

New mouse monoclonal anti–human Bcl2 and TRAIL-R1, -R2, -R3, and -R4 conjugates

What they are
The Molecular Probes® portfolio of over 1,100 highly specific primary antibodies for flow cytometry is expanding to include more Research Use Only (RUO) selections. The mouse anti–human Bcl2 antibody and the mouse anti–human TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 antibodies are now available as conjugates to expand your options for studying apoptosis.

What they offer

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

How they work
The mouse monoclonal antibody clone Bcl2/100 recognizes human Bcl2, a 26 kDa proto-oncogene with anti-apoptotic effects, expressed in the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors are transmembrane cytokine receptors. Life Technologies now offers antibody conjugates that recognize the following TRAIL receptor family members: TRAIL-R1 (CD261, DR4), TRAIL-R2 (CD262), TRAIL-R3 (CD263), and TRAIL-R4 (CD264).

Flow cytometry analysis of TRAIL-R1 expression on the surface of hematopoietic cell lines. Cells were probed with purified anti–TRAIL-R1 antibodies followed by goat anti–mouse IgG PE. Histograms represent goat anti–mouse IgG PE (no primary antibody, filled red), anti–TRAIL-R1 comparative antibody (black), and anti–TRAIL-R1 (DR-4-02) in duplicate staining (blue and green).

Over 1,500 recombinant proteins just added

Sino Biological® recombinant proteins brought to you by Life Technologies

What they are
Life Technologies is partnering with a proteomics technologies leader, Sino Biological, to deliver powerful tools to help you answer critical research questions.

For researchers working with mammalian model systems, recombinant proteins formed by mammalian expression systems are more likely to have the expected posttranslational modifications, to fold correctly, and to function properly in those models. Sino Biological uses mammalian expression systems to generate recombinant proteins that are as close to the native form as can currently be produced. Life Technologies, through a partnership with Sino Biological, plans to offer more than 2,000 recombinant proteins, many produced in mammalian expression systems. The first collection includes 150 unique viral proteins, 500 growth factors and receptors, 400 membrane-associated proteins, 180 enzymes, and 250 other cellular proteins. Life Technologies will be providing hundreds more Sino Biological® proteins in the coming months.

What they offer

  • Affordability—choose the quantity and price that meet your needs and budget
  • Activity—pure and tested for biological performance
  • Relevance—a growing list of proteins produced in mammalian expression systems

How they work
The Sino Biological® proteins offered by Life Technologies have a number of different tag options, including His, Fc, GST, His-Avi, or no tag, allowing you to tailor the activity, measurement, purification, etc., of the protein. The recombinant human proteins are supplied either as lyophilized powder or frozen liquid in four standard sizes: 5 µg, 25 µg, 50 µg, and 250 µg. If your research requires large volumes of protein or custom production, please contact us to see how the Life Technologies partnership with Sino Biological can work for you.

New fluorescent microsphere reference beads for your flow cytometer

Cell Sorting Set-up Beads and AlignFlow™ Flow Cytometry Alignment Beads

What they are
AlignFlow™ Flow Cytometry Alignment Beads are reliable references for aligning, focusing, and calibrating flow cytometers. They are available for blue, red, and UV lasers.

Cell Sorting Set-up Beads are reliable standards for flow cytometry sorter set-up and calibration. The beads are optimized for UV, blue, green/yellow, and red lasers.

What they offer

  • Consistency—uniform production creates superb reference standards
  • Confidence—help ensure the reliability of daily instrument performance
  • Compatibility—use with any flow cytometer

How they work
The AlignFlow™ beads and the Cell Sorting Set-Up Beads are microspheres infused with fluorescent dyes. They permit alignment and set-up without wasting valuable and sensitive experimental material.

Both bead types approximate the size, emission wavelength, and intensity of many labeled biological samples. The AlignFlow™ beads are highly uniform with respect to size, resulting in low forward scatter (FSC) CV and intensity CV. The Cell Sorting Set-Up Beads are optimized for examining settings such as drop delay and efficiency (cell loss during sorting).


Excitation and emission spectra for the Cell Sorting Set-up Beads for Blue Lasers. The emission spectrum was obtained for beads excited by a 488 nm blue laser.


Cell counting in color—EVOS® FL Auto Imaging System

The EVOS® FL Auto Imaging System is a fully automated, digital inverted multichannel fluorescence and transmitted-light imaging system with outstanding workflow efficiency. Designed to meet demanding requirements over a broad range of applications, it supports high-resolution mosaic tiling, multi-position well scanning, cell counting with thresholding, and time-lapse studies.

Whether you follow the step-by-step guide or manually define acquisition parameters, the EVOS® FL Auto Imaging System is remarkably easy to use and run; the touch screen display enables “finger swipes” to expand or zoom in on images, and moving between sample positions is done by a single touch.

Among the versatile software features is cell counting that can be done automatically with a captured or live image. A powerful watershed algorithm has enhanced the precision of counting cells stained with NucBlue® Live ReadyProbes™ reagent or those expressing GFP, permitting the easy determination of total cell numbers by NucBlue® Live staining, or monitoring GFP expression patterns in cell populations.

The cell-counting algorithm evaluates the signal intensity of each image pixel and combines that information with a user-defined sensitivity threshold to mark and count regions of interest (ROI; typically cells or nuclei) on the screen. The result of the count (i.e., the number of ROI above the signal threshold) is displayed, and the sensitivity can be adjusted with an on-screen slider switch, so that more or fewer ROI are tagged and counted. There is also an option that allows cells that are touching to be counted separately, increasing the accuracy of the count. The image can be saved with all the labels, counts, and percentages displayed, or with only the ROI tags showing.

Automated cell counting with the EVOS® FL Auto Imaging System.
Cell counting using the watershed algorithm makes it easy to monitor GFP expression (shown), assess live or dead cell numbers, and determine total cell count using NucBlue® Live ReadyProbes™ reagent.


Probes for neuronal tracing

Neuronal morphology and connectivity are key components in neuronal function. Molecular Probes® labeling and detection tools include a diverse selection of probes for dissecting neural networks and their functions, with an extensive palette of fluorescent colors for each class of probes.

The technologies used to trace neural networks can be divided into those for tracing single cells and those for monitoring cell populations. Although most of these probes are effective with live-tissue sections and cultured cells, some probes have been developed to address the special requirements of fixed tissues and in vivo imaging.

Use the selection guides for neuronal tracing products to compare product features and review citations from other users.



Purkinje cell and climbing fiber. Confocal image stack of a 10,000 MW Calcium Green™ dextran–labeled climbing fiber in a sagittal cerebellar slice, showing incoming axon and terminal arborization (in yellow). The Purkinje cell innervated by this fiber was labeled with Alexa Fluor® 568 hydrazide.


On the web

Secondary detection strategy guide

ProbesOnline™ readers—people who know a lot about fluorescence imaging—are often faced with complex samples in which they need to resolve a variety of targets. It takes a complete suite of fluorescent probes to detect high-, medium-, and low-abundance targets within the same sample, and to distinguish each target using wavelengths appropriate for the available excitation sources and detection channels.

Life Technologies has assembled a new web tool to help you navigate through the various options and select the right amplification level to match any target abundance.

Imaging corner

Visualizing the time course of apoptosis and cell death using NucGreen™ Dead 488 ReadyProbes™ reagent

Time course of apoptosis in HeLa cells. HeLa cells were treated with 0.5 µM staurosporine to induce apoptosis. NucGreen™ Dead 488 ReadyProbes™ reagent was applied to cells using 2 drops per milliliter of complete medium; the dye remained in the sample throughout the course of the experiment. Differential interference contrast (DIC) and green-fluorescence channel images were collected over 6 hours using a DeltaVision® Core microscope with a 40x objective; images are gain- and exposure-matched. For more information about NucGreen™ Dead 488 and other ReadyProbes™ reagents, read the BioProbes® 69 article “ReadyProbes™ ready-to-use imaging reagents for brilliant results: Simply add and image”.

Highlight from BioProbes® Journal

Oxidative stress from a cell’s point of view: CellROX® live-cell ROS sensors, and Image-iT® and Click-iT® lipid peroxidation detection kits

In the Cell Signaling section of BioProbes 69 Journal, the article “Oxidative stress from a cell’s point of view” describes three easy-to-use products for measuring various forms of oxidative stress by fluorescence microscopy, quantitative fluorescence microscopy (i.e., high-content imaging), automated microplate fluorometry, and flow cytometry:

  • CellROX® Green, CellROX® Orange, and CellROX® Deep Red reagents—fluorogenic indicators for the direct detection and quantitation of reactive oxygen species (ROS) in live cells
  • Image-iT® Lipid Peroxidation Kit—provides a simple ratiometric method for detecting the oxidative degradation of cellular lipids in live cells (see figure)
  • Click-iT® Lipid Peroxidation Kit—allows you to detect lipid peroxidation–derived protein modifications in fixed cells using click chemistry

In addition to the stand-alone CellROX® reagents, our new CellROX® Flow Cytometry Assay Kits provide validated sets of reagents for distinguishing oxidatively stressed cells, nonstressed cells, and dead cells using flow cytometry. The CellROX® Flow Cytometry Assay Kits contain reagents that have been formulated to work well together and to exhibit minimal overlap with fluorophores excited by other laser lines for multiplex experiments.

  Quantitation of lipid peroxidation in live cells using the Image-iT® Lipid Peroxidation Kit. Human osteosarcoma (U2OS) cells were plated on 35 mm glass-bottom dishes (MatTek) and stained with 10 μM BODIPY® 581/591 C11 (provided in the Image-iT® Lipid Peroxidation Kit) for 30 min in complete growth medium at 37°C. Where specified, cells were pretreated with 150 μM α-tocopherol for 30 min. Cells were then treated with vehicle (DMSO), 100 μM menadione, 200 μM tert-butyl hydroperoxide (TBHP), or 200 μM cumene hydroperoxide (CH) for 2 hr at 37°C; all cells were stained with Hoechst® 33342 stain during the last 30 min of compound incubation. The cells were then washed three times with PBS and imaged on a Zeiss® Axiovert® inverted microscope using a 40x objective and appropriate optical filters. The signal intensity was quantitated at 510 nm and 590 nm using SlideBook™ 5.0 software. In control cells, low levels of lipid peroxidation are indicated by a relatively high 590/510 ratio, reflecting predominance of the reduced dye. With treatment with menadione, TBHP, and CH, the 590/510 ratios decreased dramatically, indicating significant oxidation of the dye and higher levels of lipid peroxidation; α-tocopherol pretreatment decreased lipid peroxidation in cells.

† 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 69 Journal take shape!

Molecular Probes® webinar series

Easy cell cycle analysis

Explore an easier and more affordable solution for cell cycle analysis

Watch the free downloadable webinar, “Easier and more affordable cell cycle analysis at your bench”, to discover a new alternative for routine cell cycle analysis and learn how to perform cell cycle data modeling at your bench.