BioProbes 75 Journal of Cell Biology Applications

[Pages:37]BIOPROBES75

JOURNAL OF CELL BIOLOGY APPLICATIONS

MAY 2017

Amplify signals for fluorescence imaging

SuperBoost kits with Alexa Fluor tyramides

ALSO FEATURING Comprehensive strategy for antibody validation Super Bright fluorescent polymer dyes for the violet laser PrimeFlow RNA assay for detecting RNA targets by flow cytometry

Congratulations to the winners

of the Cell-ebrate Science Imaging Contest

We asked scientists in Singapore, Taiwan, and the Southeast Asia region to send us cell images showcasing their research using InvitrogenTM fluorescent reagents, and the submissions were stunning. After careful review by a panel of Thermo Fisher Scientific researchers, all experts in the field of fluorescence microscopy, three winning images were chosen.

Congratulations to our first, second, and third place winners for their brilliant "beautiful science" images.

1st prize winner Jaron Liu, Institute of Medical Biology, Singapore

Filopodia-like protrusions in N1E-115 neuroblastoma cells. The nanoscale architecture of filopodialike protrusions in N1E-115 neuroblastoma cells was investigated using InvitrogenTM Alexa FluorTM 568 Phalloidin (pseudocolored blue-green) and state-of-the-art super-resolution microscopy. The image was acquired on a DeltaVision OMXTM 3D-SIM with 100x 1.4NA lens, 3 ?m stack, 125 nm per stack, maximum projection, and an image pixel size of 37.5 nm.

2nd prize winner Chan Jia Pei, Duke-NUS Medical School, Singapore

Psychedelic cerebrum. A coronal section of a mouse brain was labeled with antibody markers for myelin (green, InvitrogenTM Alexa FluorTM 488 secondary antibody), axons (red, InvitrogenTM Alexa FluorTM 555 secondary antibody), and microglia (magenta, InvitrogenTM Alexa FluorTM 633 secondary antibody); nuclei were stained with HoeschtTM 33342 dye (blue). The fluorescent markers highlight the structure of the cortex, hippocampus, striatum, thalamus, and hypothalamus in this section.

3rd prize winner Jonathan Aow, National University of Singapore, Singapore

GFP-expressing primary hippocampal neuron. Primary hippocampal neurons were cultured for 15 days, transfected with GFP (green), and then fixed and stained 2 days later. Axons were labeled with anti?NF-L antibody and Alexa FluorTM 405 secondary antibody (blue), and the somato dendritic compartment was labeled with anti-MAP2A/B antibody and InvitrogenTM Alexa FluorTM 647 secondary antibody (red); neighboring untransfected neurons are visible. Images were acquired on a ZeissTM LSM700 confocal microscope with a 63x objective.

Explore our imaging resources at cellimaging

For Research Use Only. Not for use in diagnostic procedures. ? 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. DeltaVision OMX is a trademark of GE Healthcare Bio-Sciences Corporation. Hoechst is a trademark of Hoechst GmbH. Zeiss is a trademark of Carl Zeiss AG Corporation.

Cover image

Filopodia-like protrusions in N1E-115 neuroblastoma cells. The nanoscale architecture of

filopodia-like protrusions in N1E-115 neuroblastoma cells was investigated using InvitrogenTM

Alexa FluorTM 568 Phalloidin (Cat. No. A12380) and state-of-the-art super-resolution

microscopy. The image was acquired on a DeltaVision OMXTM 3D-SIM with 100x 1.4NA lens,

3 ?m stack, 125 nm per stack, maximum projection, and an image pixel size of 37.5 nm.

Image contributed by Jaron Liu, Institute of Medical Biology, Singapore.

Previous issues

BIOPROBES 74

This issue focuses on CRISPRCas9?based research, including the combination of genome editing and functional proteomics as well as a description of our comprehensive portfolio of reagents for Cas9 delivery through cell function assays. Also highlighted are several stem cell studies and fluorescence-based tools for flow cytometry and imaging, such as the FluxORTM II Green K+ channel assay.

BIOPROBES 73

This issue features Click-iTTM EdU and TUNEL colorimetric assays for immunohistochemistry, as well as SuperclonalTM recombinant secondary antibodies. Also discussed are high-speed cell counting and generational tracing assays by flow cytometry, the TurboLucTM Luciferase One-Step Glow Assay, and the SureCastTM Handcast and NovexTM WedgeWellTM precast protein gel systems.

NOVEMBER 2015

BIOPROBES 72

MOLECULAR PROBES JOURNAL OF CELL BIOLOGY APPLICATIONS

SPECIAL IMAGING ISSUE

BIOPROBES 72

In this issue, we describe

Visualize apoptosis in context

with Click-iT Plus TUNEL assays

hypoxia research tools, 3D cancer spheroid studies, and the multiplexable Click-iTTM Plus

ALSO FEATURING Intracellular detection of hypoxia Formation of uniform 3D cancer spheroids Protection from photobleaching for live cells

TUNEL apoptosis assays, as well as the CellInsightTM CX7 High-

Content Analysis Platform. We also highlight ProLongTM

Live Antifade Reagent for live-cell imaging, and

ProLongTM Diamond and SlowFadeTM Diamond Antifade

Mountants for fixed-cell imaging.

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Thermo Fisher Scientific

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For Research Use Only. Not for use in diagnostic procedures.

? 2017 Thermo Fisher Scientific Inc. All rights reserved. The trademarks mentioned herein are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. BD Horizon and Brilliant Violet are trademarks of Becton, Dickinson and Company. Cell-Mate3D is a trademark of BRTI Life Sciences Inc. cOmplete is a trademark of Roche Diagnostics GMBH. DeltaVision OMX is a trademark of GE Healthcare Bio-Sciences Corp. FLICA is a trademark of Immunchemistry Technologies LLC. GraphPad Prism is a trademark of GraphPad Software, Inc. Hoechst is a trademark of Hoechst GmbH. Luminex is a trademark of Luminex Corp. Nikon and Eclipse are trademarks of Nikon Instruments Inc. PhiPhiLux is a trademark of Oncolmmunin Inc. SIGMAFAST is a trademark of Sigma-Aldrich Co. Sprague Dawley is a trademark of Envigo. Taxol is a trademark of Bristol-Myers Squibb Company. Triton is a trademark of The Dow Chemical Company or an affiliated company of Dow. TSA is a trademark of PerkinElmer Life Sciences, Inc. VioBlue and VioGreen are trademarks of Miltenyi Biotec GmbH. Zeiss is a trademark of Carl Zeiss AG Corp.

COL03855 0517

bioprobes

Thermo Fisher Scientific 4401 E Marginal Way S Seattle, WA 98134

Antibodies seeking partners for binding relationships

Our antibodies and your antigens should get together

Over 74,000 functionally tested InvitrogenTM antibodies, covering 85% of the proteome, ready to bond. Don't be shy, meet your match--it's guaranteed.*

* Terms and conditions apply. For complete details, go to antibody-performance-guarantee

Find out more at antibodies

For Research Use Only. Not for use in diagnostic procedures. ? 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

BioProbes 75

Editors Michelle Spence Grace Richter

Designer Kim McGovern Lynn Soderberg

Production Manager Beth Browne

Contributors

Parinaz Aliahmad

Scott Brush1

Laura Allred

Yi Wen Chai1

Brian Almond

Caitlin Johnson1

Kris Barnette

Eu Han Lee1

Dan Beacham

Beth Lindborg1

Alex Behling

Joy Bickle Lisa Birkby

1BRTI Life Sciences, Two Harbors, Minnesota

Jolene Bradford

Beth Browne

John Bucci

Suzanne Buck

Gayle Buller

Wayne Considine

Jeff Croissant

William Dietrich

Melanie Dowd

Helen Fleisig

Leigh Foster

Castle Funatake

Jane Helmer

Kamran Jamil

William Kang

Kevin Kepple

Steve Lai

Hong Van Le

Dylan Malayter

Jeffrey Monette

Monica O'Hara-Noonan

Bhavin Patel

Greg Potts

John Rogers

Patricia Sardina

Laura Shapiro

Matt Slater

Dariusz Stepniak

Marcy Wickett

Michelle Yan

Zhen Yang

bioprobes?May 2017

ONLINE AND ON THE MOVE 2 | Behind the Bench blog, Molecular Probes School of Fluorescence, Antibodies Learning Center, mass spectrometry resources, and more

JUST RELEASED 4 | Our newest cellular analysis products and technologies

TOOLS FOR FLUORESCENCE IMAGING 7 |Amplify signals for fluorescence imaging SuperBoost kits with Alexa Fluor tyramides for signal amplification 11 | Cancer biology in the third dimension Fluorescent reagents for staining 3D cell cultures created with Cell-Mate3D matrix

RESEARCH WITH ANTIBODIES 13 | Harness immune checkpoints to combat tumors

Immune checkpoint antibodies for flow cytometry, IHC, and functional bioassays 16 | Comprehensive strategy for antibody validation

Characterization of antibody performance using immunoprecipitation and mass spectrometry 19 | Examine signaling pathways with targeted proteomics Quantitative analysis of the AKT/mTOR pathway using multiplex immunoprecipitation and targeted mass spectrometry

TOOLS FOR FLOW CYTOMETRY 22 | Bright fluorescent polymer dyes for the violet laser

Super Bright antibody conjugates 25 | Powering up drug discovery

Cancer biology applications using the Attune NxT Flow Cytometer 28 | Evaluate both RNA and protein targets in single cells

PrimeFlow RNA assay for detecting RNA targets by flow cytometry

JOURNAL CLUB 30 | Coming 'round to spheroid culture 32 | Measurement and characterization of apoptosis by flow cytometry

CENTER INSERT | Fluorophore and reagent selection guide for flow cytometry

Published by Thermo Fisher Scientific Inc. ? 2017

BioProbes Journal, available in print and online at bioprobes, is dedicated to providing researchers with the very latest information about cell biology products and their applications. For a complete list of our products, along with extensive descriptions and literature references, please see our website.

ONLINE AND ON THE MOVE

Strengthening our flow cytometry portfolio: eBioscience antibodies

At Thermo Fisher Scientific, we are committed to furthering your scientific advances by providing a comprehensive suite of products for the analysis of cells and their functions. The addition of the eBioscienceTM portfolio of flow cytometry antibodies and reagents to the innovative InvitrogenTM flow cytometry reagents and instruments enables us to better provide the tools your experiments demand. We are fully committed to maintaining product quality while also making the transition as seamless as possible for you. Beyond our products, we also provide dedicated service and support teams and educational resources to help support your studies. From antibodies and functional reagents to instrumentation, Thermo Fisher Scientific is now poised to more fully serve your research needs. We are focused on advancing meaningful discoveries and partnering to make tools for cellular analysis effective, affordable, and widely accessible for all life scientists. To learn more about our wide-ranging array of flow cytometry products, visit flowcytometry.

BioProbes 75

Behind the Bench blog goes with the flow

Do you work with a flow cytometer? Do you want to learn more from experienced scientists in the field? Thermo Fisher Scientific now has an archive of flow cytometry articles available on the "Behind the Bench" blog. Learn about "Flow cytometer fluidics and why they are not all the same" from Greg Kaduchak, PhD, Thermo Fisher Scientific R&D Engineer. Studying cell proliferation using CFSE or BrdU? We have the blog for you! Get advice from Andrew Filby, PhD, Flow Cytometry Core Facility Director and ISAC SRL-Emerging Leader at Newcastle University, UK, or Bill Telford, PhD, Flow Cytometry Research Core Manager at the National Cancer Institute, NIH, Bethesda, Maryland, to help you get the most from your CFSE- or BrdUbased cell proliferation assays. Read all about it at flowblog.

eLearning modules for protein and cell analysis

We are pleased to announce the release of several eLearning modules for protein and cell analysis applications. Available in the new and improved Protein and Cell Analysis Education virtual environment, these self-paced animated courses include knowledge checks throughout and a practical applications component to help you see what you have learned. The first courses cover:

T cell stimulation and proliferation: Part 1 and Part 2 Protein sample preparation--Techniques and applications: Part 1 and Part 2

All content is available 24 hours a day, 7 days a week, and is viewable from the convenience of your desk, tablet, or mobile device. See what it's all about at elearningcourses.

2 |bioprobes

? 2017 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.

BioProbes 75

ONLINE AND ON THE MOVE

The newest Molecular Probes School of Fluorescence module: Fluorescence Basics

The module "Fluorescence Basics" is now available online at the Molecular Probes School of Fluorescence. It covers the fundamentals of fluorescence, from the process of fluorescence and photobleaching to the anatomy of a fluorescence spectrum and how filters work, and is brought to you by the scientists who created the Molecular Probes Handbook and the "Imaging Basics" module. Check it out today at mpsf-fluorescence.

Antibodies Learning Center now online

InvitrogenTM primary and secondary antibodies represent a large and ever-improving portfolio of products designed to help researchers identify, locate, quantitate, and purify proteins and other biomolecules. Selecting individual antibody products from among hundreds of options can be a challenge. The Thermo Fisher Scientific Antibodies Learning Center contains educational materials designed to empower researchers and technicians with the background information about immunotechnologies that is necessary to develop, select, and use antibodies to advance their research. Learn more at ablearning.

Protein purification tools poster available upon request

Now available upon request, the protein purification tools poster provides helpful information about the Thermo ScientificTM and InvitrogenTM portfolios of magnetic beads and magnetic agarose, as well as Superflow and POROSTM resins for applications ranging from small-scale high-throughput screening to process-scale purification. This poster features practical information, including binding capacity, maximum flow rates, and the recommended application scale and packaging options for each product. Key upstream and downstream products are also highlighted as part of the integrated protein purification workflows. Request your free poster today (see terms and conditions) at purificationposter.

Mass spectrometry technical handbook now available

The Mass Spectrometry Resources Center provides valuable tools to help with your mass spectrometry experiments, including a free 166-page technical handbook, Protein sample preparation and quantitation for mass spectrometry. In addition, you'll have access to helpful white papers and late-breaking posters for specific applications like subcellular fractionation, crosslinking, peptide fractionation, isobaric labeling, and more. You can also hear from our experts on sample preparation and targeted proteomics workflows through our on-demand webinars. Register now at msresources.

? 2017 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.

bioprobes | 3

JUST RELEASED

BioProbes 75

Enhanced Alexa Fluor secondary antibodies: Higher signal-to-noise ratios for hard-to-detect targets

InvitrogenTM Alexa FluorTM Plus secondary antibodies are designed to help you detect low-abundance targets in rare or critical samples using fluorescence imaging or western blotting. The Alexa Fluor antibodies you rely on for superior brightness and photostability are now available in an exclusive formulation using proprietary "plus" dye chemistry, which provides up to 4 times higher signal-to-noise ratios. This unique formulation, coupled with stringent pre-adsorption protocols to remove antibodies that bind nonspecifically, enables Alexa Fluor Plus antibodies to offer enhanced sensitivity and minimal cross-reactivity for those challenging samples. See our complete line of Alexa Fluor Plus secondary antibodies at alexafluorplus.

Improved signal-to-noise ratios with Alexa Fluor Plus antibodies reveal greater detail. Fixed and permeabilized E18 Sprague DawleyTM primary cortical neuronal cells were labeled with anti?-III tubulin primary antibody and (A) InvitrogenTM Alexa FluorTM 488 or (B) InvitrogenTM Alexa FluorTM Plus 488 (Cat. No. A32723) secondary antibodies. Cells were then counterstained with HoechstTM 33342 dye (Cat. No. H3570) and imaged on a ZeissTM LSM 710 inverted confocal microscope at 40x magnification.

Improved formulation for Pierce protease and phosphatase inhibitor tablets

Thermo ScientificTM PierceTM protease and phosphatase inhibitor tablets are ideal for the protection of proteins during extraction procedures or lysate preparation using primary cells, cultured mammalian cells, animal tissues, plant tissues, yeast cells, or bacterial cells. With improved formulations, these ready-to-use inhibitor tablets dissolve quickly into a clear solution and are directly compatible with Thermo ScientificTM PierceTM BCA Protein Assays. Find out more at inhibitorcocktails.

Product PierceTM Protease Inhibitor Mini Tablets PierceTM Protease Inhibitor Tablets PierceTM Protease Inhibitor Mini Tablets, EDTA-free PierceTM Protease Inhibitor Tablets, EDTA-free PierceTM Phosphatase Inhibitor Mini Tablets PierceTM Protease and Phosphatase Inhibitor Mini Tablets PierceTM Protease and Phosphatase Inhibitor Mini Tablets, EDTA-free

Quantity 30 tablets 20 tablets 30 tablets 20 tablets 20 tablets 20 tablets 20 tablets

Cat. No. A32953 A32963 A32955 A32965 A32957 A32959 A32961

120 Protease inhibitors: Thermo Scienti c Roche Sigma

100

80

% Inhibition

60 40

20

0 With EDTA EDTA-free

Trypsin protease

With EDTA EDTA-free

Cysteine protease

With EDTA EDTA-free

Metalloproteases and cathepsins

Performance comparison between three commercially available protease inhibitor tablets. Pancreatic extract (100 L, 0.5 g/L) was incubated with cleavable fluorogenic substrates for trypsin and cysteine proteases, metalloproteases, and cathepsins, in the presence of Thermo ScientificTM PierceTM Protease Inhibitor Mini Tablets (Cat. No. A32953), Roche cOmpleteTM Protease Inhibitor Tablets, and SigmaAldrich SIGMAFASTTM Protease Inhibitor Cocktail Tablets, with and without EDTA. Reactions were incubated for 1 hr at 37?C, and fluorescence was determined at the appropriate detection emissions on a Thermo ScientificTM VarioskanTM Flash microplate reader. Percent protease inhibition is shown for each protease inhibitor formulation.

New and improved Tris-glycine midi protein gels

InvitrogenTM NovexTM Tris-Glycine Plus Midi Gels are the next generation of precast midi polyacrylamide gels, providing high-quality performance and separation of proteins into well- resolved bands using Laemmli electrophoresis technology. Available in fixed (10%, 12%) and gradient (4?12%, 4?20%, 8?16%) concentrations and with different well formats (12+2 wells, 20 wells, 26wells), these Tris-glycine gels feature a longer shelf life (up to 1 year at 4?C), fast run conditions ( ................
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