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Overview
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Overview

The capabilities of flow cytometry instruments in terms of available lasers, detectors and number of measurable parameters have expanded tremendously over the years. Concomitant with that, there is an exponential increase in the number of fluorochromes available for flow cytometry, extending the limits of possibilities for new discoveries. Conventional flow cytometry collects only a discrete portion of the emission spectrum as defined by a single filter per fluorochrome, whereas spectral flow cytometry collects the full spectrum. This increases the flexibility and the capability of spectral flow by enabling autofluorescence profiling and extraction, detection of more fluorochromes per laser line and increased panel size. Find out how spectral flow cytometry differs from conventional flow cytometry and how to design your panels for spectral flow cytometry.
 

Read the blog article on how spectral flow cytometry has changed the landscape of fluorochrome development for high-dimensional research.

 
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Spectral vs conventional flow cytometry comparison
 

FeatureSpectralConventional
Detector and fluorochrome ratioMore detectors than fluorochromes1:1
Individual fluorochrome contribution determinationUnmixingCompensation
Fluorochrome choiceDependent on laser configurationDependent on laser and filter configuration
Autofluorescence extractionYesNo
Resolution of highly similar fluorochromesYesNo
Multi-color panel capability40+ colors~28 colors


What remains the same?

 

  • Workflow (sample prep, staining, acquisition)
  • Need for accurate single-stain controls for unmixing
  • Panel design principles

Introduction to spectral flow cytometry

Watch the spectral flow cytometry videos below to explore various topics, such as the fundamentals of spectral and conventional flow cytometry, how you can use more fluorochromes per laser line using spectral flow cytometry, how to use spectral flow cytometry for increased panel size, and more. 

Fundamentals of spectral versus conventional flow cytometry

 

The video addresses the following questions:

  • What is spectral flow cytometry?
  • What are the similarities and differences between conventional and spectral flow cytometry?
  • How do you read spectral signatures?
  • What is unique about spectral flow cytometry?

Autofluorescence unmixing

 

The video addresses the following questions:

  • What is autofluorescence unmixing?
  • How can autofluorescence impact biological resolution?
  • What are cell-specific autofluorescence signatures?

Fluorochrome choice flexibility

 

The video addresses the following questions:

  • How to select fluorochromes in either conventional or spectral flow cytometry?
  • How does spectral flow cytometry support the use of more fluorochromes per laser line?
  • What are the advantages of using spectral flow cytometry for fluorescent protein detection?

 

Increased panel size

 

The video addresses the following questions:

  • How does spectral flow cytometry enable resolution of highly similar fluorochromes?
  • What are the similarity index and guidelines for the use of highly similar fluorochromes?
  • What are the specifics behind a 40-color multicolor spectral flow cytometry panel?
Panel Design
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Tools and considerations for the design of multicolor spectral flow cytometry

Spectral flow cytometry is an established technology that has increased the power, flexibility and complexity of flow cytometry. This webinar addresses the fundamental similarities and differences between conventional and spectral flow cytometry and dives deeper into facts and misconceptions around the principles and tools for multicolor spectral flow cytometry panel design.

Watch the webinar

 

Topics:

  • What is spectral flow cytometry and what makes it unique
  • Fundamental rules and tools for panel design common to conventional and spectral flow cytometry
  • The do’s and don’ts of panel design specific to spectral flow cytometry

BD RealBlue™ Reagent Family

Knowing which fluorochrome to use for each marker in your panel and assigning them in a way that can lead to deeper biological insights can sometimes be difficult. BD Biosciences offers new panel design resources to help with these choices as well as a wide array of antibody specificities and fluorochromes for flexible and easy panel design. Meet two of the newest additions to the BD RealBlue™ Reagent family: RB 705 and RB 744. Both RB 705 and RB 744 leverage an innovative laser-specific bright fluorochrome, to support the detection of low-expression surface and intracellular markers. RB 705 is a superior alternative to PerCP-Cy5.5 and BD Horizon Brilliant™ Blue 700 (BB700) Reagents; while RB 744 represents a bright new option for high-parameter and spectral flow cytometry panels.
 

BD Solutions
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BD Biosciences offers cell analyzers and sorters that support both conventional and spectral flow cytometry

 

What is Spectral Cytometry

Spectral flow cytometry represents an alternative data acquisition and analysis strategy to conventional flow cytometry providing advantages in flexibility of fluorescent label inputs without having to change filters, as well as the ability to multiplex more fluorescent labels in one multicolor sample.

Increase your experimental capabilities

 

Image-enabled cell sorting & advanced spectral flow cytometry

The BD FACSDiscover™ S8 Cell Sorter combines spectral flow cytometry and real-time spatial and morphological insights, significantly expanding the power of cell analysis and sorting. Marking the first spectral flow sorter with sort-capable image analysis, the BD FACSDiscover™ S8 Cell Sorter opens up new pathways to scientific discovery, enabling access to information and insights that weren’t previously attainable.

 

Learn more in the BD FACSDiscover™ S8 Cell Sorter brochure.

Plan & manage your spectral experiment

 

BD® Research Cloud is a cloud-based software solution designed to integrate and facilitate flow cytometry experiments. Its flexible workflow functionality allows labs to create customized workflows for tracking collaborative experiments, and keep all the data, metadata and resources associated with an experiment in a single-site collaborative platform. With BD® Research Cloud , you can design your next flow cytometry panel on conventional or BD spectral cytometers; minimize the time spent moving data between systems; assign workflow steps and collaborate within the platform; manage your reagent inventory, titers and master mixes; and more. BD® Research Cloud  integrates directly with FlowJo™ Software.

Build your panel with innovative dyes

 

BD Biosciences offers new panel design resources to help with these choices as well as a wide array of antibody specificities and fluorochromes for flexible and easy panel design.

Apply your spectral panel & collect data

The BD FACSDiscover™ S8 Cell Sorter features BD CellView™ Image Technology and BD SpectralFX™ Technology, which expands panel size and flexibility with 78 fluorescent detectors across five lasers with algorithmically optimized bandpass filters. Insights on cell populations and characteristics can be visually confirmed in real time during analysis and sorting. Built-in visual inspection capabilities, more flexible panel design and automated features help to simplify your workflow, supported with correlation of downstream analysis with imaging and traditional flow data. Incorporating a high level of biosafety, the BD FACSDiscover™ S8 Cell Sorter was developed in partnership with The Baker Company.

 
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The BD FACSymphony™ A5 SE Cell Analyzer features five fixed-alignment lasers and 48 detectors for maximum coverage of the fluorochrome emission spectrum. By enabling both spectral unmixing and compensation-based workflows, the BD FACSymphony™ A5 SE Cell Analyzer increases flexibility in fluorochrome choices and permits simultaneous analysis of fluorochromes with similar spectral signatures. Spectral unmixing can also be used to resolve critical cell populations using autofluorescence unmixing.

 
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The BD FACSymphony™ S6 Cell Sorter builds on the foundations of the BD FACSymphony™ A5 Cell Analyzer with easy migration of optimized panels from analyzer to sorter. The BD FACSymphony™ S6 Cell Sorter supports both conventional compensation and spectral unmixing methods, with flexibility for up to nine spatially separated lasers and 50 parameters. Together with bioinformatics analysis tools and the broad portfolio of BD high-parameter reagents, the BD FACSymphony™ S6 Cell Sorter provides an end-to-end solution for high-parameter experimental success.

 
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Analyze

Featuring an intuitive interface, specialized analysis platforms and open-ended plugin architecture, FlowJo™ Software provides a powerful environment for single-cell flow cytometry analysis that can be used for immunophenotyping, cell cycle, proliferation, kinetics studies, quantitative population comparison or plate screening assays. Analyze any data file from any cytometer, using the intuitive drag-and-drop analysis and tools to analyze high-parameter data, and generate publication-quality graphics, all with FlowJo™ Software.

 
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Educational Webinars
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Real-time image feature extraction enables a range of novel flow cytometry cell sorting applications

 

Aaron Middlebrook, Sr. Staff Scientist, BD, will highlight real-time imaging (RTI)–enabled cell sorting applications, experimental design and panel considerations.

  • Introduce you to RTI flow cytometry
  • Demonstrate how to gate and sort with quantitative image features in the same way you do with flow parameters
  • Illustrate how to apply image features for different applications to measure the relative location of fluorescence signals and track morphological changes in cell populations

   

   

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

Alexa Fluor (AF) is a trademark of Thermo Fisher Scientific. Cy is a trademark of Global Life Sciences Germany GmbH or an affiliate doing business as Cytiva.