ProbesOnline™ Newsletter

In this issue

FEATURED NEW PRODUCTS

microRNA Assays   Accurately quantitate microRNA—Qubit® and Quant-iT™ microRNA assays
     
EVOS® FLoid® Cell Imaging Station software update   Fluorescence imaging made easy and enjoyable—New and improved features in the EVOS® FLoid® Cell Imaging Station software update
     
antibodies for glutathione S-transferase (GST)   ABfinity™ recombinant antibodies—New antibodies for glutathione S-transferase (GST)
     
monoclonal anti–human CD9, anti–mouse CD9, and anti–human CD15 conjugates   Antibody conjugates for detecting CD9 and CD15 by flow cytometry—New monoclonal anti–human CD9, anti–mouse CD9, and anti–human CD15 conjugates
     
Exosome Spin Columns   Remove unincorporated dye from labeled exosomes—Exosome Spin Columns (MW 3,000)

NEW APPLICATIONS

proliferation of neural stem cells Evaluation of proliferation of neural stem cells in vitro and in vivo

PROVEN PERFORMERS

Microbial analysis using flow cytometry   Microbial analysis using flow cytometry

FEATURED NEW PRODUCTS

Accurately quantitate microRNA

Qubit® and Quant-iT™ microRNA assays

What they are
The Qubit® and Quant-iT™ microRNA Assay Kits allow easy and accurate quantification of microRNA (miRNA), even in the presence of common contaminants such as salts, free nucleotides, solvents, detergents, and proteins. The fluorescent dye–based assay is highly selective for miRNA over rRNA or large mRNAs (>1,000 bases). The assay accurately detects as little as 0.5 ng miRNA and has a dynamic range of 5–500 ng/mL (1–500 ng) in the core assay and as little as 2.5 ng/mL (0.5 ng) in the extended assay.


What they offer

  • 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 measure the fluorescence to determine the concentration

How they work
The kits provide concentrated assay reagent, dilution buffer, and prediluted miRNA standards. To perform the assay, simply dilute the reagent using the buffer provided, add your sample (any volume between 1 μL and 20 μL is acceptable), and determine the concentration using the Qubit® 2.0 Fluorometer (Qubit® assay, if you have 1–20 samples) or a fluorescence microplate reader (Quant-iT™ assay, if you have 20–2,000 samples).

Note that Qubit® microRNA assays require a Qubit® 2.0 Fluorometer, which must have the free MyQubit microRNA Assay .qbt file installed. Links to that file and easy-to-follow PDF instructions for downloading and installing it are on our Qubit® Fluorometric Quantitation web page, under “Qubit® Fluorometer Resources”.

 

 

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 Qubit® microRNA (miRNA) assay, the Qubit® RNA assay, or by 260 nm absorbance on the NanoDrop® spectrophotometer.


Fluorescence imaging made easy and enjoyable

New and improved features in the EVOS® FLoid® Cell Imaging Station software update

What it is
A new version of the software for the FLoid® Cell Imaging Station (software update V22809; FLoid® Software version 1.3) lets you save and print the exact image that appears on the screen, allows printing on either the Canon® CP800 or CP900 printer, and permits on-screen zoom-in capture by simply double-clicking or by using the zoom scale at the top left of the image.


What it offers

  • Save and Print—you can save and print the exact image you see on the screen, including the scale bar and magnified image
  • Print driver—allows you to print on both Canon® CP800 or CP900 printers
  • Image manipulation—on-screen zoom-in capture by double-clicking or by using the zoom scale

How it works
Version 1.3 improves the performance of your FLoid® Cell Imaging Station and includes bug fixes and refinements of existing features. In addition, we’ve added some frequently requested features that significantly enhance image acquisition, handling, analysis, and printing (e.g., TIFF image saving capability has been added, and system stability issues have been addressed). The software can be downloaded by clicking on the “Software & Firmware Updates” link on the FLoid® web page.

FLoid® Cell Imaging Station  

 

The FLoid® Cell Imaging Station.

 

ABfinity™ recombinant antibodies

New antibodies for glutathione S-transferase (GST)

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.

Glutathione S-transferases (GSTs) are a family of proteins present in eukaryotes and prokaryotes, where they catalyze a variety of reactions. A gene for a ~26 kDa glutathione S-transferase is part of the GST gene fusion system, which can be used to produce GST fused to almost any protein of interest. GST can act as a tag on the resulting fusion protein to help identify specific protein–protein interactions, and GST fusion proteins can be purified from cells by exploiting GST’s high affinity for glutathione. The GST gene fusion system has been introduced into numerous expression vectors.


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.

New antibodies for glutathione S-transferase (GST)  

 

Western blot of glutathione S-transferase (GST) identified with ABfinity™ GST recombinant rabbit monoclonal antibody. ABfinity™ GST recombinant rabbit monoclonal antibody (5 µg/mL) was used to label GST (5 ng) on a western blot. The western detection was performed using the WesternBreeze® Chromogenic Kit—Anti-Rabbit with NBT/BCIP as the substrate.


Antibody conjugates for detecting CD9 and CD15 by flow cytometry

New monoclonal anti–human CD9, anti–mouse CD9, and anti–human CD15 conjugates

What they are
We are expanding our portfolio of over 1,400 highly specific Molecular Probes® primary antibodies for flow cytometry to include more Research Use Only (RUO) selections. The mouse anti–human CD9, rat anti–mouse CD9, and mouse anti–human CD15 antibodies are now available as conjugates to expand your immunophenotyping options.


What they offer

  • Trust—the trusted 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 anti–human CD9 monoclonal antibodies (clones SN4 C3-3A2 and MEM-61) bind to human CD9, a transmembrane protein expressed on platelets, monocytes, pre-B lymphocytes, granulocytes, and activated T lymphocytes.

The rat anti–mouse CD9 monoclonal antibodies (clones EM-04 and KMC8) recognize the mouse CD9 antigen, also a transmembrane protein expressed on platelets, monocytes, pre-B lymphocytes, granulocytes, and activated T lymphocytes.

The mouse anti–human CD15 monoclonal antibodies (clones MEM-158 and HI98) bind to CD15, a cell membrane molecule expressed by granulocytes.

New monoclonal anti–human CD9, anti–mouse CD9, and anti–human CD15 conjugates  

 

Staining of normal human peripheral blood cells with fluorescein-labeled isotype control (blue) or mouse anti–human CD15 mAb (clone HI98), fluorescein conjugate (purple). Cells in the granulocyte gate were used for analysis.


Remove unincorporated dye from labeled exosomes

Exosome Spin Columns (MW 3000)

What they are
Exosome Spin Columns (MW 3000) enable fast removal of unincorporated dye from exosome labeling reactions. They also allow you to perform buffer exchange or to remove low molecular weight (≤3,000 MW) components from the exosome preparation, including salts, nucleotides, and short oligonucleotides.


What they offer

  • Fast, efficient removal of unincorporated dye from labeled exosomes
  • Buffer exchange, desalting, and removal of low molecular weight contaminants
  • Replaces more time-consuming separation methods (such as ultracentrifugation)

How they work
To use, simply rehydrate a spin column, centrifuge to remove the interstitial fluid, add the exosome sample to the top of the column, and centrifuge again to elute the exosomes. Low molecular weight contaminants will be retained by the column. 

Exosome Spin Columns
Uptake by HeLa cells of exosomes labeled with SYTO® RNASelect™ stain. A FLoid® Cell Imaging Station was used. Red: Alexa Fluor® 594 phalloidin; blue: DAPI; green: SYTO® RNASelect™ stain. Exosomes are 30–150 nm extracellular vesicles believed to be present in all body fluids. They have been shown to transport cargo (proteins, lipids, and various types of RNA) from one cell to another, and are thought to play a role in a large number of biological functions.

NEW APPLICATIONS

Evaluation of proliferation of neural stem cells in vitro and in vivo

Morte MI, Carreira BP, Machado V et al. (2013) Curr Protoc Stem Cell Biol Chapter 2: Unit 2D.14.

Studies that need to evaluate growth of neural stem cells (NSCs), in culture or in tissues, have frequently used incorporation of the thymidine analog 5-bromo-2´-deoxyuridine (BrdU) during DNA synthesis as a measure of cell proliferation. However, because BrdU incorporation is detected using an antibody, cells must undergo harsh treatment to denature the DNA (typically with hydrochloric acid or heat) prior to antibody addition. Those treatments can cause the loss of other epitopes and damage tissue structure, making additional study of the sample problematic.

Morte et al. have recently published detailed methods for evaluation of NSC proliferation that do not require DNA denaturation. Instead of BrdU, they used 5-ethynyl-2´-deoxyuridine (EdU), also a thymidine analog that is incorporated into DNA during synthesis. Rather than antibody detection, EdU is detected using “click” chemistry. EdU, which contains an alkyne, can undergo a copper-catalyzed reaction that causes the alkyne and a fluorescent dye–labeled azide to form a stable covalent bond. The small size of the azide reagents means that the fluorescent label has efficient access to the EdU in the DNA without the need for denaturation.

In one of the protocols included in the publication, Morte et al. combine detection of EdU incorporation to measure NSC proliferation, with cell cycle analysis using the DNA-binding dye 7-aminoactinomycin D (7-AAD). In another protocol they describe a method to colocalize EdU incorporation with the simultaneous labeling of NSC maturation markers, both internal and on the cell surfaces. In both of these protocols, proliferation is measured by EdU incorporation detected using click chemistry, because the DNA denaturation step required for detecting BrdU incorporation would make the other labeling procedures difficult or impossible.

Life Technologies has developed Click-iT® EdU Assay Kits optimized for flow cytometry, imaging, high-content screening, and high-throughput screening applications, and we also provide EdU products and guidelines for use in whole animals. In addition to better accommodating multiplex labeling, EdU methods are simpler and faster than BrdU methods. From start to finish, our optimized click chemistry assay for EdU detection is complete in as little as 90 minutes, whereas the antibody-based BrdU method takes 6 to 24 hours to complete.

Flow cytometric analysis of cell proliferation using Click-iT® technology  

 

Flow cytometric analysis of cell proliferation using Click-iT® technology. Jurkat cells were treated with 10 µM EdU for 2 hr, then fixed and permeabilized, labeled via the click reaction, washed, and counterstained for cell cycle analysis using SYTOX® AADvanced™ stain. Cells were analyzed using the Attune® Acoustic Focusing Cytometer equipped with 405 nm and 488 nm excitation and 450/50 nm bandpass and 675/20 nm bandpass filters.

PROVEN PERFORMERS

Microbial analysis using flow cytometry

With its capacity to analyze large numbers of cells in a short amount of time, flow cytometry is particularly suitable for environmental microbiology and microbial physiology studies. Flow cytometry has proven useful for quantitating both viable and unculturable microorganisms, for studying host–microbe interactions, and for measuring different facets of microbial metabolism.

Life Technologies offers a family of BacLight™ products specific for microbiology studies using flow cytometry. The BacLight™ Green and BacLight™ Red Bacterial Stains are compatible with both gram-positive and gram-negative bacteria, and live, alcohol-fixed, and formaldehyde-fixed bacteria all show bright fluorescence after staining. These bacterial stains can be combined with other fluorescent probes, including nucleic acid stains, lectin conjugates, and antibody conjugates, for multiparameter analyses.

The BacLight™ RedoxSensor™ Green Vitality Kit provides a nontoxic fluorogenic indicator of bacterial reductase activity, as well as the nucleic acid stain propidium iodide and two different electron transport disruptors (sodium azide and CCCP) to produce controls. BacLight™ RedoxSensor™ Green Indicator exhibits bright green fluorescence when modified by bacterial reductases, many of which are located in the electron transport system. BacLight™ RedoxSensor™ Green Indicator has been used to detect bacteria in sediment samples and also to separate bacteria capable of methane metabolism from other cells during fluorescence-activated cell sorting (FACS) (Kalyuzhnaya MG, Lidstrom ME, Chistoserdova L (2008) ISME J 2, 696–706). In this study, BacLight™ RedoxSensor™ Green Indicator was pivotal in the isolation of a Methylobacter species responsible for methane metabolism; although previously detected using culture-independent methods, this particular species was not culturable until after sorting. As shown below, actively metabolizing Escherichia coli cells stained with BacLight™ RedoxSensor™ Green Indicator exhibit bright fluorescence when analyzed using the Attune® Acoustic Focusing Cytometer.

Microbial analysis using flow cytometry
Analysis of relative cell vitality in a bacterial culture using flow cytometry. (A) Escherichia coli cells were left untreated or treated with an electron transport chain uncoupler (sodium azide), stained with the BacLight™ RedoxSensor™ Green Vitality Kit, and analyzed using the Attune® Acoustic Focusing Cytometer equipped with the blue (488 nm) laser. Samples were run at the standard collection rate of 25 µL/min, and fluorescence emission was detected using a 530/30 nm bandpass filter. The fluorescence histogram overlay indicates that untreated cells show brighter green fluorescence—corresponding to greater bacterial reductase activity—than do cells treated with sodium azide. (B) E. coli cells were grown in either nutrient-rich LB broth or nutrient-poor minimal medium, stained with the BacLight™ RedoxSensor™ Green Vitality Kit, and analyzed using the Attune® Acoustic Focusing Cytometer. The fluorescence histogram overlay indicates greater redox potential in cells grown in the rich LB broth (green), compared with cells grown in nutrient-poor minimal medium (blue); unstained cells represented in red.

DEPARTMENTS

On the web

   

Product selection guides for cell tracing, tracking, and morphology

Molecular Probes® fluorescent products comprise a unique toolbox for studies of cell proliferation, migration, chemotaxis, and invasion. We’ve built a web page where you can explore labeling strategies that range from expressing fluorescent proteins in the target cells to applying cell-permeant cytoplasmic labels. The page also includes links to multiple options for neuronal tracing.

We offer a diverse range of products that label cells in a variety of ways to enable you to view changes in morphology and location. Trackers and tracers in a wide assortment of colors also provide efficient and sensitive methods for monitoring specific cells within a population by flow cytometry or imaging, in culture or in whole animals.

Find links to selection guides on our cell tracing, tracking, and morphology page

   

“Exosomes—The next small thing”: A mini-documentary series

We asked 10 prominent scientists to share their thoughts—on camera—on the exciting field of exosome research. Based on these interviews, we are producing a six-part mini-documentary series that will tell the history and story of this new field and its impact on research areas such as cancer and immunology. The series will also give an overview of potential future therapeutic and diagnostic applications that may come from exosome research. Whether you’re into exosomes or not, this is a must-see!

Imaging corner

Z-stack image series of a tubulin-, mitochondria-, and nuclear-labeled HeLa cell

 

HeLa cells were transduced with CellLight® Tubulin-GFP and CellLight® Mitochondria-RFP and incubated overnight. NucBlue® Live ReadyProbes™ Reagent was added the next day, and three series of Z-stack images were acquired at 0.366 µm step sizes using the DAPI, GFP, and RFP filters on the EVOS® FL Auto Imaging System. The three Z-stack series were merged and are shown here in filmstrip form but can also be viewed as a movie.

Blebbing of the cell membrane, which can indicate loss of cell health, can be seen at the top end of the Z-stack in the GFP channel, with tubulin apparently present in the membrane blebs. The EVOS® FL Auto Imaging System’s Z-stack capabilities enabled the capture of this phenomenon; it would not have been visualized in a widefield, three-color, flat-focus image.

Highlight from BioProbes® Journal

Automated imaging. Simplified: The EVOS® FL Auto Imaging System.

In the Cell Imaging section of BioProbes 69, the article “Automated imaging. Simplified.” describes the new EVOS® FL Auto Imaging System. With automated stage movement, filter and objective changes, focus, and exposure, this multichannel fluorescence imaging system gives you access to high-level imaging applications, including:

  • Time-lapse acquisition
  • Multi-well plate scanning
  • Image stitching and tiling
  • Automated cell counting

When equipped with the optional EVOS® Onstage Incubator, the EVOS® FL Auto Imaging System allows you to acquire high-resolution time-lapse movies spanning many hours in a precisely controlled stagetop environment. The EVOS® Onstage Incubator is fully integrated with the imaging system and operated by the same software and user interface, enabling you to regulate temperature, humidity, and three different gas levels for the duration of your experiment.

  Time-lapse series of HeLa cells undergoing cell division. HeLa cells were transduced with CellLight® Histone 2B-GFP and CellLight® Mitochondria-RFP. Following an overnight incubation, cells were imaged every 30 min for 16 hr on the EVOS® FL Auto Imaging System using a 20x objective and the EVOS® Onstage Incubator.

† 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 take shape!

Life Technologies University

Upcoming flow cytometry workshops

Comprehensive flow cytometry and advanced applications workshop (FLO-105)
October 21–25
Frederick, Maryland, USA

This 5-day course is a combination of both lecture and wet lab experience. Mornings will consist of a seminar presentation and roundtable discussion of topics including basic theory and principles of flow cytometry, fluorescence compensation, data interpretation, multicolor panel design, and the vast array of applications for which flow cytometric technology can be used. Following lunch, participants perform practical exercises in the lab and learn techniques including sample preparation, instrument operation, data acquisition, and analysis of common flow cytometry applications: viability, cell cycle, T cell isolation and characterization, immunophenotyping, and apoptosis.