In this issue


ReadyProbes™ Nuclear Stains   Nuclear staining has never been easier—ReadyProbes® nuclear stains for live and fixed cells
Alexa Fluor® Amine-Reactive Labels   No better time to react—Alexa Fluor® amine-reactive labels
  Keep your cells healthy during imaging, and see the results in time lapse—EVOS® Onstage Incubator for EVOS® FL Auto Imaging System
CellROX® Flow Cytometry Assay Kits   Detect reactive oxygen species (ROS) in live cells using flow cytometry—CellROX® Flow Cytometry Assay Kits
Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B   Visualize autophagosome-to-autolysosome progression in live cells—Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B
SiteClick™ Antibody Labeling Kits   Site-specific antibody labeling in 3 easy steps—SiteClick™ Antibody Labeling Kits
ReadyProbes™   Ready-to-use Alexa Fluor® dye–conjugated secondary antibodies—ReadyProbes® fluorescent antibodies in convenient dropper bottles
  Accurate microRNA quantitation—MyQubit miRNA Assay
Tali® Cell Cycle Kit   Quick and easy cell cycle analysis at your benchtop—Tali® Cell Cycle Kit
LysoTracker® Deep Red Reagent   Near-infrared lysosomal dye—LysoTracker® Deep Red


antibodies for flow cytometry   Qdot® nanocrystals conjugated to antibodies for flow cytometry—CD2, CD19, CD4, and CD45R primary antibodies
ABfinity™ recombinant antibodies™ Bioparticles® Conjugates   Antibodies engineered for consistency—ABfinity™ recombinant antibodies



BioProbes® Journal of Cell Biology Applications

The Molecular Probes® Handbook



Nuclear staining has never been easier

ReadyProbes® nuclear stains for live and fixed cells

What they are
Cell-permeant and -impermeant nuclear stains in ReadyProbes® format are available in multiple colors. All emit bright fluorescence when bound to DNA and are formulated in a room temperature–stable solution and come in convenient dropper bottles. Two drops per milliliter of medium is all it takes to stain your cells.

What they offer

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

How they work
The cell-permeant NucBlue® and NucRed® Live reagents stain DNA and nuclei of both live and fixed cells and can be used for counterstaining, DNA content, and total cell counting. The membrane-impermeant NucBlue®, NucGreen®, and NucRed® Dead stains easily stain cells with compromised cell membranes, yet don’t enter living cells. They are ideal for discrimination of live and dead cells, and staining of fixed cells and tissue slices. Finally, propidium iodide is a classic dead-cell marker for flow cytometry applications, and we are pleased to make it available in ReadyProbes® format.

ReadyProbes Nucelar Stains   Rapid staining of dead cells using NucGreen® Dead stain. HeLa cells were treated with 0.5 µM staurosporine to induce apoptosis and cell death and stained with NucGreen® Dead 488 ReadyProbes® Reagent (2 drops of reagent per milliliter of medium). Overlaid DIC and green channel images were collected over a 6 hr period using a DeltaVision® Core microscope with a 40x objective. Images are gain- and exposure-matched.

Time course of cell death visualized using ReadyProbes® reagents. 
A549 cells grown in a MatTek glass-bottom dish were stained with both NucRed® Dead 647 ReadyProbes® Reagent and NucGreen® Dead 488 ReadyProbes® Reagent by adding 2 drops of the reagents per milliliter of complete medium and incubating for 20 min prior to disrupting membrane integrity by addition of DMSO/Triton® X-100 surfactant. The time-lapse video was acquired using a 40x objective on a DeltaVision® Core microscope using DIC, green, and far-red filter channels to show green nuclear staining in damaged cells.

No better time to react

Alexa Fluor® amine-reactive labels

What they are
You can conjugate these amine-reactive reagents with virtually any protein or peptide for subsequent use in fluorescence microscopy, high-content screening, and flow cytometry applications.

Different packaging sizes of the popular Alexa Fluor® amine-reactive dyes offer added convenience for performing small- or large-scale conjugations. The 3 x 100 µg size is ideal for optimizing reaction conditions, because it provides Alexa Fluor® amine-reactive dyes in single-use quantities, eliminating the need to perform multiple conjugations simultaneously or re-aliquot a larger unit size and risk losing material in the process. For large-scale reactions, the larger unit size eliminates the need to combine multiple vials.

What they offer

  • Convenience—focus on the reaction, not dye handling, with optimized unit sizes
  • Brightness—superior fluorescence intensity compared to other spectrally similar fluorophores
  • Photostability—more time for image capture

How they work
Alexa Fluor® dyes are reactive molecules that can be used to add a fluorescent label to primary amines of proteins, amine-modified oligonucleotides, and other amine-containing molecules.

Alexa Fluor® Amine-Reactive Labels
Detecting stem cell surface markers using fluorescently labeled IgM antibodies.
Tra-1-81 antibodies were labeled using (A) Alexa Fluor® 488 SDP ester, (B) Alexa Fluor® 594 NHS ester, and (C) Alexa Fluor® 647 NHS ester. H9 human embryonic stem cells on a feeder layer of irradiated murine embryonic fibroblasts were cultured on hESC medium containing 20% KSR and 4 ng/mL bFGF. Cells were stained with dye-conjugated antibodies at 5 µg/mL, and images were collected on a Zeiss Axiovert® fluorescence microscope at 10x magnification.

Keep your cells healthy during imaging, and see the results in time lapse




EVOS® Onstage Incubator for EVOS® FL Auto Imaging System

What it is

The EVOS® Onstage Incubator is an environmental chamber designed specifically for the EVOS® FL Auto Imaging System. The environmental chamber fits on the automated X-Y stage. A small, separate control unit supplies the power and gas (air, CO2, and N2 for hypoxia experiments), and controls humidity and temperature. Several interchangeable vessel holders are available and make time-lapse imaging easy and convenient.

What it offers

  • Easily controls a fully integrated environmental chamber for live-cell time-lapse imaging
  • Precisely maintains physiological or nonphysiological conditions
  • Intuitively sets all environmental and image acquisition parameters from the EVOS® FL Auto interface

How it works
Together, the EVOS® FL Auto system and the EVOS® Onstage Incubator enable precise control of temperature, humidity, and three gases for time-lapse imaging of live cells under both physiological and nonphysiological (e.g., hypoxia) conditions. Environmental settings and image acquisition parameters are all seamlessly integrated into the EVOS® FL Auto interface, creating a high-performance inverted imaging system with unmatched flexibility, ease of use, and superb optical performance for demanding live-cell time-lapse imaging experiments.

Detect reactive oxygen species (ROS) in live cells using flow cytometry

CellROX® Flow Cytometry Assay Kits

What they are
We now offer three Molecular Probes® kits for oxidative stress detection via flow cytometry. The kits feature CellROX® reagents, probes that become brightly fluorescent when oxidized. Included in each kit is one of the CellROX® reagents formulated for use in flow cytometry, a color-matched dead-cell stain to distinguish dead cells from oxidatively stressed and nonstressed live cells, an antioxidant (N-acetylcysteine [NAC], to serve as a negative control), and an oxidant (tert-butyl hydroperoxide [TBHP], to serve as a positive control).

What they offer

  • Fluorogenic probes—CellROX® reagents become brightly fluorescent when oxidized in cells
  • Multicolor compatibility—minimal overlap with fluorophores excited by other laser lines, allowing easy multiplexing with other reagents
  • Simple—cells can be stained in complete media or other appropriate buffers; no need for serum-free media
  • Fixable—CellROX® Green and CellROX® Deep Red Reagents retain signal following formaldehyde fixation (CellROX® Orange Reagent is not compatible with fixation)

How they work
The three CellROX® reagents all detect ROS in live cells. Each reagent is cell-permeant and is nonfluorescent or very weakly fluorescent in the reduced state. Upon oxidation, the reagents exhibit strong fluorescence and remain localized within the cell. When used together with the SYTOX® Red Dead Cell Stain (or SYTOX® Blue Dead Cell Stain with CellROX® Deep Red Reagent), oxidatively stressed and nonstressed cells are reliably distinguished from dead cells by flow cytometry.

Reactive oxygen species (ROS) detected by flow cytometry 
Reactive oxygen species (ROS) detected by flow cytometry. (A)
ROS levels detected by the CellROX® Deep Red Reagent are decreased in TBHP-treated Jurkat cells with pretreatment of cultures using NAC. The cells treated with the oxidant TBHP (red) have increased staining with the CellROX® Deep Red Reagent, compared to the cells pretreated with NAC (blue) and the control cells (green). (B, C) CellROX® Deep Red Reagent can be used in conjunction with SYTOX® Blue Dead Cell Stain to differentiate live stressed cells from dead cells. Jurkat cells were treated with (B) PBS or (C) 200 μM TBHP for 30 minutes before labeling with the CellROX® Deep Red Flow Cytometry Assay Kit. Note that the treated cells (C) have a higher percentage of cells under oxidative stress than the basal level of ROS observed in control cells (B).

Visualize autophagosome-to-autolysosome progression in live cells

Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B

What it is
The Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B is a fluorescent protein–based biosensor that reports the formation of the autophagosome, followed by the maturation of the autophagosome as the lysosome fuses to it to form the autolysosome. This biosensor provides a more in-depth investigation of autophagy in live cells, over time.

What it offers

  • More in-depth autophagy data—monitor the maturation of the autophagosome into the autolysosome
  • A complete solution—use in conjunction with LysoTracker® Deep Red to monitor the complete pathway of autophagy
  • Highly efficient—BacMam 2.0 technology enables autophagy to be studied across a broad range of cell types, including primary human cells, neurons, and stem cells

How it works
The Premo™ Autophagy Tandem Sensor combines the autophagy marker LC3B with two fluorescent proteins, GFP and tagRFP. GFP is sensitive to low pH, whereas tagRFP is not. At the neutral pH of the autophagosome, fluorescence is observed from both GFP and tagRFP. Once the lysosome has fused, the pH drops, quenching GFP fluorescence and leaving only tagRFP fluorescence.

Premo™ Autophagy Tandem Sensor
Mechanism of action of the Premo™ Autophagy Tandem Sensor. (A)
Schematic representation of BacMam-mediated gene delivery and expression of the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B. (B) Validation of the Premo™ Autophagy Tandem Sensor as a biosensor to report changes in pH in LC3B-positive compartments. Treatment of cells with the vehicle causes no detectable accumulation of autophagosomes, resulting in a diffuse pattern of GFP and RFP fluorescence. Inhibition of autophagy using either chloroquine or leupeptin A results in the accumulation of LC3B-positive vesicles (autophagosomes/autolysosomes). Chloroquine neutralizes the pH of lysosomes, and therefore GFP and RFP fluorescence emission is observed. Leupeptin A doesn’t affect lysosomal pH, so only fluorescence from tagRFP is observed.

Site-specific antibody labeling in 3 easy steps

SiteClick™ Antibody Labeling Kits

What they are
The SiteClick™ Antibody Labeling Kits allow you to conjugate your IgG antibodies to Qdot® particles (525, 565, 585, or 605 nm emission) or DIBO‑modified R-phycoerythrin (R-PE).

What they offer

  • Simplicity—in 3 simple steps, expertly attach Qdot® particles or R-PE to your antibody
  • Quality conjugates—enzymatic labeling system keeps antibodies and antigen-recognition site intact

How they work
In the first step of SiteClick™ conjugation, the Fc region of the antibody is modified by β-galactosidase. The azide-containing sugar UDP-GalNAz is then added to the modified carbohydrate domain of the antibody via the β-1,4-galactosyltransferase (GalT)-catalyzed reaction targeting the specific N-linked oligosaccharide GlcNAc residues. This specific targeting maintains the integrity of the antigen-binding site on the antibody. Finally, the antibody (now containing an azide moiety) is conjugated to the DIBO-modified label (Qdot® particle or R-PE) in a copper-less click reaction with a simple overnight incubation.

SiteClick™ conjugation reaction
Click to enlarge
SiteClick™ conjugation reaction.

Ready-to-use Alexa Fluor® dye–conjugated secondary antibodies

ReadyProbes® fluorescent antibodies in convenient dropper bottles

What they are
Our Alexa Fluor® dye–conjugated secondary antibodies for immunocytochemistry are bright and often play an important role in published research. We took the brightest and most frequently used conjugates and put them into a ReadyProbes® ready-to-use format. Supplied in dropper bottles, ReadyProbes® reagents can eliminate the need to pipet solutions, make calculations, or prepare dilutions when you stain cells.

What they offer

  • Brilliant Alexa Fluor® secondary antibody conjugates, ready to use in dropper bottles
  • No dilutions, pipetting, or optimization required; just add, wash, and detect
  • Plenty of options for multiplexing

How they work
ReadyProbes® Alexa Fluor® dye–conjugated antibodies are supplied ready to use, in dropper bottles. After incubation with the primary antibody is complete, simply add 2 drops of ReadyProbes® antibody per milliliter of sample, incubate, wash, and image. For multiplexing, you can choose ReadyProbes® formulations of either Alexa Fluor® 488 or 594 dye conjugated to anti–mouse IgG or anti–rabbit IgG antibodies that were raised in donkey or goat hosts.

ReadyProbes™ fluorescent antibodies  
Multiplex cell labeling using ReadyProbes® fixation and staining reagents. Caki (human kidney carcinoma) cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit and then stained. Nuclei are stained with NucBlue® Fixed Cell ReadyProbes® Reagent (blue), actin of the cytoskeleton is stained with ActinGreen™ 488 ReadyProbes® Reagent (green), and mitochondria are labeled with mouse anti–ATP synthase subunit IF1 monoclonal antibody and detected with ReadyProbes® Alexa Fluor® 594 goat anti–mouse IgG antibody (red).

Accurate microRNA quantitation

MyQubit miRNA Assay

What it is
The MyQubit miRNA Assay for use with the Qubit® 2.0 Fluorometer allows easy and accurate quantitation of small amounts of miRNA even in the presence of ribosomal RNA, using a combination of existing Life Technologies reagents, common buffers, and the MyQubit miRNA Assay file. The MyQubit miRNA Assay is based on the Quant-iT™ OliGreen® ssDNA Reagent, which exhibits a large increase in fluorescence upon binding to nucleic acids.

What it offers

  • Sensitivity—accurately detect as little as 0.5 ng miRNA, even in the presence of ribosomal RNA
  • Simplicity—add your sample (in any volume between 1 μL and 20 μL), then read the concentration using the Qubit® 2.0 Fluorometer

How it works
The MyQubit miRNA Assay is selective for small RNA, unlike other RNA detection methods such as the NanoDrop® UV-VIS spectrophotometer A260 assay or even the Qubit® RNA assays (see figure). The MyQubit miRNA Assay has a dynamic range of 5–500 ng/mL miRNA. The MyQubit miRNA Assay file can be downloaded from the bottom of the Qubit® 2.0 Fluorometer web page and permanently uploaded to your Qubit® 2.0 Fluorometer. In addition to the MyQubit miRNA Assay, the MyQubit firmware preloaded on all new Qubit® 2.0 instruments allows you to create other assays for your Qubit® 2.0 Fluorometer. Because the instrument is operated by simple commands, creating additional applications can be as straightforward as matching the spectral characteristics of the assay with the right LEDs and emission filters.

Comparison of detection techniques for accurate quantitation of small RNA in the presence of ribosomal RNA. rRNA at the concentrations listed was spiked into solutions containing 2 ng/µL siRNA, then read using the MyQubit miRNA assay, the Qubit® RNA assay, or by 260 nm absorbance (A260) on the NanoDrop® spectrophotometer.

Quick and easy cell cycle analysis at your benchtop

Tali® Cell Cycle Kit

What it is
The Tali® Cell Cycle Kit provides a room temperature–stable, ready-to-use solution for convenient and accurate determination of the percentage of cells in each phase of the cell cycle. This kit contains a single solution composed of propidium iodide (PI), RNase A, and Triton® X-100 in buffer at optimized concentrations.

What it offers

  • An all-in-one solution—contains everything you need to stain cells for cell cycle analysis
  • Simple protocol—fix your cells, add reagent, incubate, and visualize
  • Flexibility in analysis of results—data can be analyzed on the Tali® instrument or easily exported for in-depth analysis using modeling software of your choice

How it works
PI is a red-fluorescent molecule that binds DNA and RNA by intercalating between the bases with little or no sequence preference; the binding is proportional to the amount of these nucleic acids, so PI can be used to accurately determine the amount of cellular DNA for cell cycle studies after treating samples with RNase. The Tali® Cell Cycle Kit includes both PI and RNase in a single solution.

Tali® Cell Cycle Kit  
Cell cycle histogram obtained using the Tali® Cell Cycle Kit and the Tali® Image-Based Cytometer. Jurkat cells were fixed with 70% ethanol, then stained with 200 µL of the Tali® Cell Cycle Kit solution. After incubation for 30 minutes, the samples were analyzed on the Tali® Image-Based Cytometer using the “Cell Cycle” assay. The resulting histogram has the expected distribution for normal cycling cells.

Near-infrared lysosomal dye

LysoTracker® Deep Red

What it is
LysoTracker® Deep Red is a red-shifted near-infrared probe designed to label lysosomes for fluorescence microscopy or flow cytometry.

What it offers

  • Selectively detects lysosomes—monitored in the Cy®5 channel with peak Ex/Em of 647/668 nm
  • Easy multiplex capability—combine with green- or red-fluorescent reagents such as GFP and RFP
  • Complete solutions—use in conjunction with the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B to monitor the complete pathway of autophagy

How it works
LysoTracker® Deep Red is a novel lysosomotropic agent that accumulates in acidic structures. In most cells these are limited to lysosomes. The excitation and emission properties of the LysoTracker® Deep Red reagent have been modified so that they exactly match the Cy®5 fluorescence channel, facilitating multiplex imaging with GFP and RFP.

LysoTracker® Deep Red Reagent
Labeling of lysosomes with LysoTracker® Deep Red. (A, B)
U2OS cells expressing CellLight® Lysosomes-GFP were labeled with 50 nM LysoTracker® Deep Red. LysoTracker® Deep Red shows colocalization with CellLight® Lyso-GFP. (C) Multicolor imaging using LysoTracker® Deep Red. HeLa cells expressing CellLight® Tubulin-GFP and CellLight® Lysosomes-RFP were labeled with Hoechst 33342 and LysoTracker® Deep Red.


Qdot® nanocrystals conjugated to antibodies for flow cytometry

CD2, CD19, CD4, and CD45R primary antibodies

What they are
Qdot® primary antibody conjugates of the mouse anti–human CD2 and CD19 markers and the rat anti–mouse CD4 and CD45R (B220) markers are available in a variety of colors for flow cytometry.

What they offer

  • Compatibility—use together with organic dye conjugates to increase the number of detectable parameters
  • Stability—nanocrystals do not degrade over time like tandem conjugates, so they afford greater reproducibility
  • Minimal single-laser compensation—narrow emission spectra allow for minimal compensation when using a single excitation source

How they work
Qdot® nanocrystal primary antibody conjugates have extremely bright fluorescence emissions that make them well suited for the detection of low-abundance cell-surface proteins. They can be efficiently excited using a 405 nm (violet) light source. In addition, the narrow, symmetric emission profiles of Qdot® conjugates require substantially lower compensation compared to conventional fluorescent conjugates, enabling better, more efficient multicolor assays using the violet laser.

Qdot® Primary Antibody Conjugates  
Histogram overlay plot of gated human lymphocytes. The black line represents cells labeled with CD2 Mouse Anti-Human mAb (clone S5.5) Qdot® 655 Conjugate, and the gray line represents unstained cells. Samples were acquired and analyzed using 405 nm excitation and a 660/20 bandpass emission filter using the Attune® Acoustic Focusing Cytometer (Blue/Violet option).

Antibodies engineered for consistency

ABfinity™ recombinant antibodies

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.

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 chain 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.

ABfinity™ recombinant antibody for Bcl-2  
Bcl-2 localization in HeLa cells. Immunocytochemistry analysis of HeLa cells probed with ABfinity™ Bcl-2 recombinant rabbit oligoclonal antibody using (A) Alexa Fluor® 488 goat anti–rabbit IgG as a secondary antibody (green), (B) DAPI to stain nuclei (blue), and (C) Alexa Fluor® 594 phalloidin to stain actin (red). (D) Composite image of the cells, showing cytoplasmic and nuclear localization of Bcl-2.


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