Resources
Instrument Configurations
Cell Sorter Configurations
Cell Analyzer Configurations
ImageStream
Instrument Guides and Standard Operating Procedures
Cell Sorter Guides and SOPs
Cell Analyzer Guides and SOPs
10x Genomics
Resources for Experimental and Panel Design
The experimental design process, including proper panel design, is critical for the success of a flow cytometry experiment. We have assembled a variety of resources to help you through these often complex steps. In addition, we are always available to provide assistance through consultations and on-instrument guidance.
Sample Preparation
The quality of your sample will determine the success of your experiment. You must ensure that the sample is prepared as a robust single-cell suspension and is debris free. You must also ensure that cell viability is maintained during preparation and acquisition or sorting.
Protocols for preparation of blood, spleen, bone marrow and other immune tissues have been well-established and standardized. However, preparation of sample from solid tissue, often requiring mechanical and enzymatic disaggregation, is application-specific and must be optimized for the type of tissue you are working with.
It is best to start with a literature search or a protocol that has been established by someone in your lab or colaborator.
For further resources:
Reichard, et.al. have prepared a highly useful resource in Cytometry Part A for preparation of single cells from solid tissue:
Miltenyi Biotec has also developed many products for gentle sample preparation.
Enzymes can be obtained from many sources. Wothington Biochemical has developed an Enzyme Manual and Tissue Dissociation Guide that are helpful in developing your protocol.
Panel Design
Panel design is the process of assigning fluorochromes to cellular markers. This process involves many factors, including compatibility of fluorochromes to the particular instrument you plan to use, fluorochrome brightness, considerations of spillover and spillover spreading, marker expression level, and autofluorescence of the target cells.
You can use the Panel Design and Optimization document as a starting point, and our staff can help you throughout the process.
Fluorofinder is a useful free online platform for panel design. All of the CSCI Flow Cytometry instruments have been uploaded to Fluorofinder. Please make sure to submit your panel for review before purchasing reagents.
Panel Design Services
In addition, antibody vendors, including Thermo Fisher and BioLegend, offer free panel design services.
- You can find out more about Thermo Fisher's resource here. To begin the panel design process with Thermo Fisher, please contact the Panel Design Team.
- Please contact Diana Vesely, Ph.D., Technical Application Scientist Lead, BioLegend, for more information about BioLegend's services.
Spectra Viewers
Spectra viewer online tools, which display the excitaton and emission spectra of commercially available fluorochromes in a user friendly format, are critical tools when designing flow cytometry experiments. You are highly encouraged to use one of the following tools when choosing reagents for any flow cytometry experiment, even those requiring only a few colors.
Bio-Rad Fluorescence Spectraviewer
FPBase (spectra viewer for fluorescent proteins)
Thermo Fisher Fluorescence SpectraViewer
Sample Preparation for Cell Sorting
The sample requirements for cell sorting are more nuanced than for flow cytometry analysis alone. We have prepared a Cell Sorting FAQ that will help guide you when preparing for a cell sorting experiment.
Basic Protocols
BioLegend's Technical Protocols page provides a multitude of protocols for flow cytometry assays, including cell surface and intracellular antigen staining.
The Thermo Fisher Flow Cytometry Protocol Handbook covers sample preparation, immune cell stimulation, phenotypic analysis, cell viability and apoptosis, cell cycle, cell proliferation.
Key Literature References
General Flow Cytometry
From the reviews of the 3rd Edition...
"The standard reference for anyone interested in understanding flow cytometry technology."
American Journal of Clinical Oncology
"...one of the most valuable of its genre and...addressed to a wide audience?written in such an attractive way, being both informative and stimulating."
Trends in Cell Biology
This reference explains the science and discusses the vast biomedical applications of quantitative analytical cytology using laser-activated detection and cell sorting. Now in its fourth edition, this text has been expanded to provide full coverage of the broad spectrum of applications in molecular biology and biotechnology today. New to this edition are chapters on automated analysis of array technologies, compensation, high-speed sorting, reporter molecules, and multiplex and apoptosis assays, along with fully updated and revised references and a list of suppliers.
Multicolor Panel Design
Maciorowski, Z., Chattopadhyay, P.K., & Jain, P. (2017). Basic multicolor flow cytometry. Current Protocols in Immunology, 117, 5.4.1– 5.4.38. doi: 10.1002/cpim.26
Mair, F., Tyznik, A.J. (2019). High-Dimensional Immunophenotyping with Fluorescence-Based Cytometry: A Practical Guidebook. In: McCoy, Jr, J. (eds) Immunophenotyping. Methods in Molecular Biology, vol 2032. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9650-6_1
Mahnke YD, Roederer M. Optimizing a multicolor immunophenotyping assay. Clin Lab Med. 2007 Sep;27(3):469-85, v. doi: 10.1016/j.cll.2007.05.002. PMID: 17658403; PMCID: PMC2034273.
The Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling Technologies: The most complete fluorescent labeling and detection reference available, The Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling Technologies contains over 3,000 reagents and kits representing a wide range of Invitrogen Molecular Probes labeling and detection products. The significantly revised 11th edition features extensive references, reorganized content, and new technical notes and product highlights.
Cell Sorting
Arnold LW, Lannigan J. Practical issues in high-speed cell sorting. Curr Protoc Cytom. 2010 Jan;Chapter 1:Unit 1.24.1-30. doi: 10.1002/0471142956.cy0124s51. PMID: 20069524.
Cell Viability, Proliferation and Apoptosis
Hollville, E. and Martin, S.J. 2016. Measuring apoptosis by microscopy and flow cytometry. Curr. Protoc. Immunol. 112: 14.38.1- 14.38.24. doi: 10.1002/0471142735.im1438s112
Johnson, S., Nguyen, V. and Coder, D. (2013), Assessment of Cell Viability. Current Protocols in Cytometry, 64: 9.2.1-9.2.26. https://doi.org/10.1002/0471142956.cy0902s64
Kim, K.H. and Sederstrom, J.M. 2015. Assaying cell cycle status using flow cytometry. Curr. Protoc. Mol. Biol. 111: 28.6.1- 28.6.11. doi: 10.1002/0471142727.mb2806s111
Lyons, A.B., Blake, S.J. and Doherty, K.V. (2013), Flow Cytometric Analysis of Cell Division by Dilution of CFSE and Related Dyes. Current Protocols in Cytometry, 64: 9.11.1-9.11.12. https://doi.org/10.1002/0471142956.cy0911s64
Pozarowski, P., Grabarek, J. and Darzynkiewicz, Z. (2003), Flow Cytometry of Apoptosis. Current Protocols in Cell Biology, 21: 18.8.1-18.8.33. https://doi.org/10.1002/0471143030.cb1808s21
User Manuals
Cell Sorters
Cell Analyzers
ImageStream
Other Instrumentation
Journals and Educational Resources
Flow Cytometry Journals
Cytometry Part A, the journal of quantitative single-cell analysis, features original research reports and reviews of innovative scientific studies employing quantitative single-cell measurement, separation, manipulation, and modeling techniques, as well as original articles on mechanisms of molecular and cellular functions obtained by cytometry techniques.
Cytometry Part B: Clinical Cytometry features original research reports, in-depth reviews and special issues that directly relate to and palpably impact clinical flow, mass and image-based cytometry. These may include clinical and translational investigations important in the diagnostic, prognostic and therapeutic management of patients.
Current Protocols in Cytometry is a comprehensive collection of protocols and review articles focused on the detection and analysis of various cellular populations, including instrumentation, data processing and analysis, cell and molecular imaging, and more.
Educational Resources
CYTO University (CYTO U), The Internal Society of the Advancement of Cytometry's comprehensive online learning portal.
OpenFlow Cytometry is a free and extremely informative educational video series.
The Flow Cytometry Core at MSKCC is an amazing resource for many facets of flow cytometry, including technical and experimental aspects.
Expert Cytometry is a flow cytometry educational company that offers an array of interactive online flow cytometry courses.
Work-Flow is an online flow cytometry educational platform with a multitude of resources to help you develop your flow cytometry skills.
Flow Cytometry Societies and Communities
The International Society for the Advancement of Cytometry (ISAC) is the preeminent global community of cytometry professionals. ISAC hosts an annual multi-day meeting, CYTO, in May or June.
MetroFlow, the NY/NJ Flow Cytometry User Group, is a local group of cytometry professionals that hosts an annual one-day meeting in October. MetroFlow has archived its talks from past meetings which can be found on it's YouTube page.
FlowTex is a flow cytometry user group based in Texas with global reach. FlowTex welcomes attendees both in-person and virtually from anywhere in the world and is a fantastic resource for all flow cytometry professionals. Follow FlowTex's YouTube channel for archived talks and other resources.