Antibody Insider—Issue 2017 Q3
Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are pluripotent stem cells (PSCs) that can self-renew and have the potential to become any cell type found in the body. PSCs can be cultured in vitro and have been used to generate various lineage cell types of different germ layers for studying developmental biology and disease mechanisms and also for cell therapy.
It becomes critical to maintain a set of cell markers specific to the various stages of differentiation to understand the quality of the culture being used. Below we list a set germ layer markers, each linked to the associated Invitrogen antibody products, which can be used to characterize the resulting cell types. In the figure below, we have used antibodies for beta-3 tubulin, smooth muscle actin and alpha-fetoprotein to identify ectoderm, mesoderm and endoderm generated from iPSCs, respectively.
In addition, we offer many Invitrogen antibodies for identification of PSCs, adult stem cells and other specific cell types. Our Stem Cell Research Antibodies page (thermofisher.com/stemcellabs) provides further information on using antibodies in stem cell research.
Representative germ layer markers
|Mesoderm||Smooth muscle actin|
Embryoid body tri-lineage staining. Embryoid bodies (EB) were generated from Gibco Human Episomal iPSCs grown on Geltrex matrix (A1413301 and A1413302) in Essential 8 Medium. After 3 weeks in culture, EB were dissociated with TrypLE reagent and re-plated onto Geltrex matrix–coated multiwell plates. Cells were fixed, permeabilized and blocked for immunostaining. Primary antibodies (beta-3 tubulin, MA1-118; SMA, MA5-11547; and AFP, MA5-14665) were added overnight at 4°C at a 1:100 dilution. After 3 washes, appropriate secondary antibodies labeled with Alexa Fluor 488 dye were added at a 1:500 dilution in conjunction with NucBlue Fixed Cell ReadyProbes Reagent. After another 3 washes, the cells were imaged on EVOS FLoid Cell Imaging Station.
The characterization of stem cells is a critical step in stem cell research. No matter which detection platform you use—flow cytometry, immunocytochemistry, western blot, ELISA, or another—our collection of Invitrogen stem cell antibodies provides you with tools compatible with your experimental design.
Selected antibodies for the characterization of stem cells and neural lineage cells. For a complete list, go to thermofisher.com/antibodies.
Representative stem cell markers
|Characterization of pluripotent stem cells|
|Pluripotent stem cells||DNMT3B|
|Germ layer mesendoderm||Brachyury (T)|
|Germ layer mesoderm||ABCA4|
|Smooth muscle actin|
|Germ layer endoderm||Alpha-fetoprotein (AFP)|
|Germ layer ectoderm||Beta-3 tubulin|
|Neural stem cells||Nestin|
|Neural differentiation and characterization of glial and neuronal cells|
|DA progenitor/DA neurons||LMX1A|
New and unique monoclonal antibodies to explore human pluripotency
Monoclonal antibodies (mAbs) are effective tools for objective characterization of various cell types. More specifically, mAbs that detect cell-surface proteins are useful because they can be employed for positive and negative selection of cell populations via techniques such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).
Historically, a select panel of mAbs has been used to characterize human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), collectively termed human pluripotent stem cells (hPSCs) [Trends Cardiovasc Med 13:295 (2003)]. This panel includes mAbs that detect the stage-specific embryonic antigens 3 and 4 (SSEA-3 and SSEA-4). SSEA-3 and SSEA-4 antibodies detect globo-series glycolipids, and are raised against mouse embryos and embryonic carcinoma cells.
Another series of antibodies raised to embryonic carcinoma cells (TRA-1-60, TRA-1-81, and GCTM-2) are associated with a pericellular matrix of keratin sulfate/chondroitin sulfate proteoglycan. Most of these antibodies are of the IgM class and comprise a pentameric polypeptide. This structure, when compared to other immunoglobulin classes, is larger, more difficult to handle and label, and typically is associated with higher levels of background staining. Hence, the availability of new well-characterized mAbs detecting cell-surface epitopes on hPSCs would provide useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming.
In response to this need, we have recently described the generation of new mAbs that detect cell-surface proteins present on primed and naive human hESCs and hiPSCs [Stem Cells 35:626 (2017)]. This confirms our previous prediction that these proteins were present on the cell surface of hPSCs [Stem Cells 27:2446 (2009)].
mAbs to four predicted proteins have now been produced and characterized. These antibodies detect CUB domain–containing protein 1 (anti-hCDCP1; CSTEM26), platelet F11 receptor (anti-hF11R; CSTEM27), desmoglein-2 (anti-hDSG2; CSTEM28), and cadherin-3 (anti-hCDH3; CSTEM29). Each of the four monoclonal antibodies detects target protein on live hPSCs by flow cytometry (see figure below). These data show a high correlation by intracellular flow cytometry for the transcription factor Oct-4.
Find CSTEM mAbs using the table below
|Clone||Target name||Format||Size||Cat. No.|
|CSTEM26||CD318 (CDCP1)||Unconjugated||25 µg||14-3189-80|
|Alexa Fluor 647||25 Tests||51-3189-41|
|CSTEM27||CD321 (F11R)||Unconjugated||25 µg||14-3219-80|
|Alexa Fluor 488||25 Tests||53-3219-41|
|CSTEM28||Desmoglein 2||Unconjugated||25 µg||14-9159-80|
|Alexa Fluor 488||25 µg||53-9159-80|
Representative flow cytometry dot plots showing high co-expression of target proteins. The mAbs detected hPSC-surface proteins (Alexa Fluor 488 dye) and Oct-4 (Invitrogen Alexa Fluor 647 dye) against isotype controls following sequential live- and fixed-cell immunolabeling of WA09 hESCs.
StemFlex Medium—designed for better everything
Gibco StemFlex Medium supports the robust expansion of feeder-free PSCs and is optimized to deliver superior performance in novel applications, including single-cell passaging, gene editing, and reprogramming. Its unique formulation offers the convenience of a flexible feeding schedule (including weekend-free options) and also the ability to choose the matrix and passaging reagent that best suits specific applications. StemFlex Medium enables the long-term feeder-free culture of PSCs without karyotypic abnormalities and maintains cells’ ability to differentiate into all three germ layers up to 50 passages.
- Superior performance in gene editing, single-cell passaging and other stressful applications
- Out-of-the-box solution with minimal optimization and no additional reagents required
- Use when you need a robust formulation for everyday culture
- Great for difficult cell lines
iBright Imaging Systems—stunningly easy western blot imaging
Researchers will now have a simpler time capturing and analyzing data from gels and western blots with the introduction of the Invitrogen iBright Imaging Systems. Designed with a streamlined, intuitive interface and workflows, iBright Imaging Systems are easy to use for researchers of all experience levels.
Two iBright Imaging Systems models are available: the iBright CL1000 and the iBright FL1000. The iBright CL1000 model is capable of imaging chemiluminescence-based western blots in addition to stained protein and nucleic acid gels. The iBright FL1000 model features the same imaging modes as the iBright CL1000, and also offers fluorescent blot imaging capability, in both visible and near-IR channels.
The iBright Imaging Systems are equipped with Smart Exposure acquisition technology for the rapid determination of optimal exposure times—minimizing the need to repeat exposures to get the desired signal. Combined with a powerful 9.1 megapixel camera, iBright Imaging Systems have robust imaging potential, helping enable the detection of subtle differences in samples. Advanced automated features such as automatic sample rotation, auto-zoom, auto-focus, and automatic on-board data analysis provide for a smooth imaging experience.
Pair Alexa Fluor and Alexa Fluor Plus secondary antibodies with the iBright FL1000 Imaging System
iBright FL1000 models feature five fluorescence channels, which enable up to four-color fluorescence western blot multiplexing. This multiplexing potential allows researchers to study multiple proteins in each blot so that more meaningful and representative blotting experiments are possible—combine what could be several blotting experiments into one high-throughput experiment.
High throughput blotting experiment with 4 mini blots captured in a single image. Proteins detected with target-specific primary antibodies and the following secondary antibodies: Alexa Fluor 488 (blue), Alexa Fluor 546 (yellow), Alexa Fluor Plus 680 (red), Alexa Fluor Plus 800 (green).
Learn more about iBright Imaging Systems or request a demo today.
Using Brilliant Violet antibody conjugates? Meet the Super Bright alternative
Discover your options with the newest violet-fluorescent Invitrogen eBioscience Super Bright antibody conjugates for flow cytometry. Now available in four formats, these antibody conjugates allow for additional channels off the violet laser while providing better discrimination of dim cell populations.
Emission spectra of Super Bright 436, Super Bright 600, Super Bright 645, and Super Bright 702 polymer dyes. The black bar indicates the excitation wavelength of the violet laser (405 nm). The less-intense, brown curve under the blue emission shows the contribution of the (donor) Super Bright 436 dye to the emission curves of the three tandem Super Bright polymer dyes.
Less ambiguity in your antibody drug conjugate (ADC) screening data
Antibody drug conjugates (ADC) represent a novel method to deliver a drug to diseased cell using the specific antigen binding capability and internalization. Only ADCs that internalize efficiently—and deliver the intended payload—warrant inclusion in subsequent screens. Our reactive chemistries, bright and versatile fluorophores, and detection platforms help ensure that your time and limited investment dollars are used to maximum benefit and that your internalization screens are efficient and deliver actionable data.
Site–specific binding with minimal disruption to your antibody
Small modifications can impact an ADC’s binding efficacy and thereby its ability to internalize. Specifically attaching a putative drug molecule to sugars on the Fc fragment using Invitrogen SiteClick conjugation chemistry helps ensure that the antibody’s specific antigen binding capacity is preserved.
See the BioProbes article: The SiteClick antibody labeling system
Using pH change to detect and measure internalization
Invitrogen iFL pHrodo dyes dramatically increase in fluorescence as pH changes from basic to acidic (see the figure below). Attachment of an iFL pHrodo dye to your antibody makes for a fast, effective and unambiguous probe for the study of antibody internalization.
Clear internalization of Herceptin (Genentech) antibody in breast cancer cells. Herceptin antibody conjugates of amine-reactive iFL pHrodo Red traffic into HER2+ SK-BR-3 cells and become brightly fluorescent in acidic lysosomes.
Efficient internalization of ADCs
The two figures below show binding and internalization of ADCs using flow cytometry and high-content image analysis.
Specific binding and internalization of Gazyva obinutuzumab (Roche) in CD20+ cells using flow cytometry. Cells from triplicate samples were analyzed on the Attune NxT Flow Cytometer with AutoSampler to show binding specificity and internalization of Gazyva obinutuzmab to CD20+ cells. Non-specific binding and internalization was not in observed in CD20– cells.
Efficient tracking of internalization events measured using high-content image analysis. Conjugated Herceptin (Genentech) antibody internalizes into Her2+ cells over time. Her2- negative cells provide minimal signal. Cells from triplicate samples were analyzed on the Thermo Scientific CellInsight CX5 Platform.
Secondary Antibody Cross-Adsorption and Cross Reactivity—new resource in the Antibodies Learning Center
Are you experiencing these types of issues with your imaging results?
- High background
- Nonspecific binding
- Multiplexing cross-reactivity with other primary and secondary antibodies
If you answered yes to either of those questions, then you should consider your secondary antibody’s level of cross-adsorption. A new article explaining cross-adsorption and how to improve your results is now available in the Antibodies Learning Center.
Castle Funatake, PhD
Senior Manager of R&D
Thermo Fisher Scientific
Do you work with a flow cytometer and want to learn more about the newest Super Bright antibody conjugates for flow cytometry? Check out the Super Bright antibody conjugate Flow Cytometry article on the Behind the Bench blog. Learn insights about these newest dyes from expert, Castle Funatake, PhD who is the leader of the R&D team that developed the Super Bright antibody conjugates.
Pluripotent Stem Cell Guidebook—recently updated
Whether you are new to the field or looking to take your stem cell research in a new direction, we have the tools and resources you need to help ensure your success. Access our new and improved Pluripotent Stem Cell Guidebook for our complete portfolio of products and services, aimed at helping you in every step of your research including stem cell culture, reprogramming, differentiation and characterization. We also have products for the more challenging parts of your workflow including transfection, gene editing, and single-cell passaging.
Download the guidebook
Immune Cell Guide for Human and Mouse Antigens—recently updated
Need to know the markers and their locations for a given immune cell type? We have the guide for you! Download the updated Immune Cell Guide as a reference for your lab today.
Download the guidebook
Introducing the Thermo Fisher Scientific education resource
With this free virtual learning platform, you can access webinars, posters, and papers about applications and techniques related to protein gel chemistries, western detection, mass spectrometry, flow cytometry, fluorescence imaging, high content imaging, antibodies, immunoassays, and other topics.
To learn more, visit thermofisher.com/proteincelledu
Webinar—What are the current advances in Flow Cytometry?
Speaker: J. Paul Robinson, PhD
Presented at: Protein and Cell Analysis Education Series
- Compare hydrodynamic and acoustic focusing in flow cytometry
- List three applications where the Attune NxT provides a unique benefit
- Describe automated plate reader options for the Attune NxT
Webinar—Antibody Validation Forum
Speakers: Panel comprising: Aled Edwards, PhD; Matt Baker; Anita Bandrowski, PhD; Paul K. Wallace, PhD; John Rogers, PhD; Christoph Hergersberg, PhD
Presented at: Protein and Cell Analysis Education Series
- Understand the nature of the antibody reproducibility crisis
- Gain insight about the guidelines developed by the International Working Group for Antibody Validation (IWGAV)
- Learn about the scientific methodologies designed to verify antibody specificity
To access all of our webinars, visit Protein and Cell Analysis Webinars On-Demand
Take an eLearning course!
Our animated, narrated courses provide succinct, contextual information about protein and cell analysis topics including protein sample preparation and purification and T cell subset phenotypic analysis. Complete the eLearning challenge and download a certificate.
Learn more at thermofisher.com/elearningcourses
Molecular Probes School of Fluorescence—Flow Cytometry Basics module
Ever wonder how a flow cytometer works? Need to understand the optical set-up of a flow cytometer? Now you can find the answers to these questions and more in the Molecular Probes School of Fluorescence—Flow Cytometry Basics module now online.
This free resource was created by our scientists to help you get started in with flow cytometry, which can be a complex and challenging application.
BioProbes 75 is now available online!
Read the latest issue of BioProbes Journal of Cell Biology Applications now available online. Antibody-related articles include:
Harness immune checkpoints to combat tumors
Immune checkpoint antibodies for flow cytometry, IHC, and functional bioassays
Comprehensive strategy for antibody validation
Characterization of antibody performance using immunoprecipitation and mass spectrometry
Examine signaling pathways with targeted proteomic
Quantitative analysis of the AKT/mTOR pathway using multiplex immunoprecipitation and targeted mass spectrometry
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