Centre for Immunology and Vaccinology CL3 Cell Sorting, Chelsea and Westminster Hospital

LMS NIHR Flow Cytometry Facility, Hammersmith

St. Mary's Flow Cytometry Core Facility

Cytometry Part A

The official journal of the International Society for Advancement of Cytometry focuses on quantitative single-cell analysis. It features original research reports and reviews of innovative scientific studies employing quantitative single-cell measurement, separation, manipulation, and modelling techniques, as well as original articles on mechanisms of molecular and cellular functions obtained by cytometry techniques.

Optimized Multicolor Immunofluorescence Panel (OMIP)

Reports on newly designed and optimized multicolour panels for flow cytometry, image cytometry and other polychromatic fluorescence-based methods. OMIPs alleviate the development time for researchers in need of the same or highly similar panels, provide a starting point for the creation of novel OMIPs, and give panel credit to the developers.

Cytometry Part B: Clinical Cytometry 

The official Journal of the International Clinical Cytometry Society 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

It is a comprehensive source for 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.

Cold Spring Harbor Protocols 

Interdisciplinary journal providing a definitive source of research methods. It does not have a dedicated section for flow cytometry. However, it allows searching for protocols for antibody screening, assessment of apoptosis, cell death measuring and others.

Books

Practical Flow Cytometry by Howard Shapiro

This is the Bible of flow cytometry. The basics of flow cytometry remain very similar over the last decades, and this is a text everyone goes back to solve even the more complex issues of the new technologies. It includes Shapiro's Eight Laws of Flow Cytometry.

Flow Cytometry - A Basic Introduction by Michael G. Ormerod

Electronic version of the book edited in 2008, provided by De Novo Software. This handbook covers flow cytometry's basis theory and deals exclusively with mammalian systems. A must-read for beginners in flow cytometry.

Mario Roederer's Compensation Perspective

An entire website dedicated to compensation: why it is important, how to do it, and the most usual errors when doing it. Also recommended is his paper “Compensation in Flow Cytometry” (doi: 10.1002/0471142956.cy0114s22).

Single Cell Analysis - Contemporary Research and Clinical Applications by J. Paul Robinson and Andrea Cossarizza

Purdue University Cytometry Laboratories manual, published by Springer in 2017. This book highlights the current state of the art in single cell analysis – from clinical hematology, functional analysis and drug screening, to platelet and microparticle analysis, marine biology and fundamental cancer research.

High-Dimensional Immunophenotyping with Fluorescence-Based Cytometry: A Practical Guidebook by Florian Mair and Aaron Tyznik 

A chapter of "Immunophenotyping: Methods and Protocols" (2019), edited by J. Philip McCoy Jr, dedicated to providing a practical step-by-step protocol for designing high-dimensional fluorescent panels and focusing on common challenges. The authors claim to successfully establish a 30-parameter panel in a 3–6 week time frame. Great for users of the Penteon and Aurora.

Flow Cytometry of Hematological Malignancies by Claudio Ortolani

ESCCA Manual, published by Wiley-Blackwell in 2011. An array of graphical outputs produced by flow cytometry in the study of neoplastic blood diseases. The images included allow you to compare your own results with a third party reference pattern. There is a detailed description of the main leukocyte antigens, together with a description of their distribution amongst normal and abnormal blood cells, and a comprehensive description of the disease phenotype.

Scientific articles

Herzenberg LA, Tung J, Moore WA, et al. Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol. 2006 Jul;7(7):681-5. doi: 10.1038/ni0706-681. - A foundational text on fluorescence-activated cell sorting that has had a great impact on improving the common practices of labs worldwide.

Maecker HT, Trotter J. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A. 2006 Sep 1;69(9):1037-42. doi: 10.1002/cyto.a.20333. - A good guide to voltration and gating controls in conventional flow cytometry.

Hulspas R, O’Gorman MR, et al. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry B Clin Cytom. 2009 Nov;76(6):355-64. doi: 10.1002/cyto.b.20485. - For those specially working on clinical samples and spectral flow cytometry, this is a good basis knowledge on autofluorescence and its causes.

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. - Tips and tricks on preparing and executing multicolour flow cytometry for immunomonitoring. Especially dedicated to users of conventional flow cytometry.

Ferrer-Font L, Pellefigues C, et al. Panel Design and Optimization for High-Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry. Curr Protoc Cytom. 2020 Mar;92(1):e70. doi: 10.1002/cpcy.70. - A foundational text for users of Aurora and other spectral flow cytometry. The authors highlight the specific characteristics of spectral flow cytometry and aim to guide users through the process of building, designing, and optimizing high-dimensional spectral flow cytometry panels using a comprehensive step-by-step protocol. 

Ferrer-Font L, Small SJ, et al. Panel Optimization for High-Dimensional Immunophenotyping Assays Using Full-Spectrum Flow Cytometry. Curr Protoc. 2021 Sep;1(9):e222. doi: 10.1002/cpz1.222. - A continuation of the previous paper, for users of spectral flow cytometry in immunophenotyping. The authors focus on optimizing a high-dimensional full-spectrum flow cytometry panel for immunophenotyping using comprehensive step-by-step protocols and video tutorials. These protocols can also be used to troubleshoot panels when issues arise.

Liechti, T., Weber, L.M., et al. An updated guide for the perplexed: cytometry in the high-dimensional era. Nat Immunol. 2021 Sep;22: 1190–7. doi: 10.1038/s41590-021-01006-z. - A comprehensive guide to navigate high-parameter cytometry data analysis, ensuring a correct and transparent pipeline for reproducible results.

Protocols

• Worthington Tissue Dissociation Guide
• The importance of area scaling with FACS DIVA software
• Combined Flow Cytometric Measurement of Two Cell-Surface Antigens and DNA-RNA Content
• Flow Cytometric Preservation Protocol (for marine and freshwater samples)
• Tips and Tricks – Analyzing Challenging Samples within a Core Laboratory

iBiology

• A Brief Introduction to Flow Cytometry
• FACS – Fluorescence Activated Cell Sorting

BD Introduction to Flow Cytometry Web-Based Training

A beginners webinars course that describes flow cytometry principles, components and function of each part of the instrument, the sorting process and how to analyse data.

OpenFlow Cytometry

An international open access Flow Cytometry education resource with comprehensive videos on:

• Conventional flow cytometry experiment setup on Diva,
• Compensation, voltration, panel design and multicolour troubleshooting on Diva,
• Cell sorting on Diva,
• Spectral flow cytometry with Cytek Aurora,
• Full spectrum panel design and troubleshooting,
• Data analysis, including DNA analysis and cell proliferation.

UChicago Flow

Playlist managed by the Flow Facility at the University of Chicago. The videos are being monetized and the revenue is sent to Chicago User Group (ChUG) Cytometry, a non-profit scientific educational group on everything flow cytometry. Some recommended videos are:

• Navigating the NovoExpress software for Penteon and performing compensation,
• Tips on using SpectroFlo for Cytek Aurora,
• Sample prep and flow workflow for single cell seq,
• FCSExpress tutorial

Flow Cytometry Training 2020 - The Francis Crick Institute

A detailed list of webinars developed by the flow cytometry department at Crick Institute during the 2020 COVID-19 lockdown. Recommended videos include:

• Light scatter tutorial,
• Fixation tutorial,
• Apoptosis detection,
• Post-sorting single cell genomics,
• Data presentation,
• Introduction to imaging flow cytometry,
• Introduction to mass cytometry

Cyto University

ISAC's learning resources for flow cytometry. It provides live webinars, peer-reviewed and recorded courses, and tutorials from CYTO conferences, among other interactive elements. Register as a fee-paying member or a free non-member.

Clinical Cytometry Education Network

CCEN hosts educational resources for the international clinical cytometry community, from practical clinical cytometry courses to training of young professionals via the Visitor Training Program.

BD Biosciences Fluorescence Spectrum Viewer - The best option for those working with BD instruments and conventional flow cytometry. Only works in a desktop browser.

Invitrogen Spectrum Viewer - More advanced than the BD's viewer, but good if you have unusual fluorophores and fluorescent proteins. Only works in a desktop browser.

Cytek Aurora Full Spectrum Viewer - The best option for those working with our Aurora and spectral flow cytometry. Only works in a desktop browser.

NovoCyte Spectra Viewer - The best option for users working with our NovoCyte Penteon. Only works in a desktop browser.

Biolegend Fluorescence Spectra Analyzer - Now with conventional and spectral cytometry analysis. Also available as App for Android.

FPbase - The Fluorescent Protein Database

A free and open-source, community-editable database for fluorescent proteins (FPs) and their properties. It is mainly directed at microscopy purposes, but it contains invaluable information on FP performance under different conditions, a collection database of available plasmids and a chart of FP properties.

For a comprehensive list of fluorescent protein brightness, visit Takara's chart.

Semrock Searchlight - A very advanced spectral viewer for flow cytometry and microscopy. Also available as App for Android.

Before attempting to build your panel, review our Blackboard course on panel design and read the following entries:

• How to identify problems with flow cytometry panel design
• Panel Design and Optimization for High-Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry
• Panel Optimization for High-Dimensional Immunophenotyping Assays Using Full-Spectrum Flow Cytometry

Planning tools

Bio-Rad planning tools - A free resource with panel design layouts, 96-well plates experiment layouts and shopping lists.

Panel builders

Chromocyte - A free resource with panel builder and antibody database, including suppliers.

FluoroFinder - An all-in-one platform that contains spectral viewers for conventional and spectral flow cytometry and a panel builder, and other features such as suppliers and an inventory option. You will need to create an account to use it.

If you prefer not to create an account, you can use the panel builder located in BioLegend's website, which is powered by FluoroFinder.

OMIPs

“Optimized Multicolor Immunofluorescence Panel” (OMIP) alleviate the development time for researchers who wish to use the same (or highly similar) panels; provide a starting point for the creation of novel OMIPs; and provide a mechanism for attribution to the developers of the panel via citation of the publication on Cytometry A.

The Luxemburg Institute of Health and the Palo Alto Veterans Institute for Research have developed the OMIP_ISAC, a dynamic tool that allows to visualise the OMIPs entries by number, marker, species, application, etc.

Antibody search engines

Antibodies-online - A platform that allows you to compare antibodies based on available validation data only, with standardised data for a better decision-making process.

FluoroFinder Product Search - An all-in-one platform with an antibody search tool that links directly to the suppliers and allows for comparisons between reagents.

BenchSci - An AI-driven antibody selection tool to plan more successful experiments. It has expanded to include cell lines, RNAi, CRISPR and others. You will need to create an account to use it.

Publishing

Minimum Information about a Flow Cytometry Experiment

MIFlowCyt promotes that the published results of any study have to be open to independent validation or refutation. It establishes criteria for recording and reporting information about the flow cytometry experiment overview, samples, instrumentation and data analysis. FlowRepository is a database of flow cytometry experiments where you can download data collected and annotated according to the MIFlowCyt standard. These data can also be used to learn how to use data analysis software. If used, reference accordingly:

• Spindler et al, 2012, in Current Protocols in Cytometry
• Spindler et al, 2012, in Cytometry Part A

To help you in the process of presenting your data, watch this webinar that discusses the guidelines available to help with robust, reliable and reproducible data presentation.

Analysis software

FlowJo

Developed by BD for the analysis of all types of cytometry data. FlowJo University is a complete set of videos to understand how to operate FlowJo to analyse single-cell phenotypes, from the basics of the layout editor to proliferation and high parameter analysis algorithms.

For more information, you can watch this series of webinars that discuss data analysis, including DNA analysis and cell proliferation.

SpectroFlo

Developed by Cytek for the analysis of spectral cytometry data. SpectroFlo Video Tutorials is a playlist that covers everything from daily maintenance of Cytek Aurora to editing the workspace and keeping an updated reference library of single-colour controls.

FCS Express

Developed by De Novo Software, it is preferred for the analysis of clinical cytometry data. FCS Express Video Resources offers recorded versions of all their webinars as well as short overviews of most features in FCS Express. They have uploaded Pipelines for HD Data Analysis a playlist to start you on high-dimensional analysis. All videos are available for direct download.

For another good tutorial, watch this FCS Express Tutorial hosted at the University of Chicago Flow Cytometry Facility.

Software Carpentry

A volunteer project dedicated to teaching basic computing skills to researchers. GitHub, Python and R are covered in their in-person workshops, and lessons are available in their website free of charge, bith in English and Spanish.

tSNE vs. UMAP

Dimension reduction techniques such as tSNE and UMAP are absolutely central for many types of big data analysis in cytometry. Learn how to interpret them and how to create them in the following links:

• Visualizing Data Using t-SNE
• t-SNE vs UMAP
• tSNE vs. UMAP: Global Structure

Catalog of Free Flow Cytometry Software

Managed by the Purdue University Cytometry Laboratories. This catalogue strives to be comprehensive -- to include as much free flow cytometry software as possible. It also includes some demonstrations of commercial packages.

Cytobank

A cloud-based platform for the analysis of big single-cell data:  Sunburst, Dose Response, Heat Maps, SPADE, viSNE, CITRUS, Dot and Histogram Overlays. It requires a license.

Flow Cytometry UK 

Formed in June 2006 to promote the interaction between all users of flow and imaging cytometry within the UK. They hold regional meetings to foster interaction between users and promote new applications and techniques. In addition, this site acts as a central resource for links, protocols, meeting advertisements and other relevant information. Their regional London Cytometry Club holds regular meetings and attendance is free.

Royal Microscopical Society 

The RMS is dedicated to furthering the science of microscopy.  It does that through a wide range of activities that support research and education in microscopy, cytometry and imaging.  The Flow Cytometry Section of RMS was established specifically to encourage communication between cytometrists in both clinical and research laboratories.

International Society for the Advancement of Cytometry

ISAC is the professional organisation for scientists utilising analytical cytometry, the multidisciplinary, advanced technology for the measurement of cells and cellular processes for biological and diagnostic purposes. It provides resources for facility staff, data standards, meeting advertisements and online education, including the Minimum Information about a Flow Cytometry Experiment and the Cyto University.

Purdue University Cytometry Laboratories

The first site web site in cytometry, created in 1993. It contains video tutorials, reference materials, protocols, software, jobs and more. It contains the much loved Cytometry Discussion List, a searchable international mailing list questions and answers for flow cytometry users.

Cytek Aurora User Forum

An online community for users of Cytek Aurora that is free to access, consult and take part in.

Clinical Flow Wiki

This wiki-format site is devoted to building a connected community around clinical flow cytometry, and to provide information, case examples, e-Learning and resources to its members.

European Society of Clinical Cell Analysis

ESCCA aims at the standardisation, validation and dissemination of flow cytometric know-how and its implementation in clinical laboratories. Aside from highly valuable educational resources, it contains the multicentre Eurostandards project (2004), which developed a number of technical guidelines on cell subset identification and absolute counting, stem cell enumeration and subsetting, micro-bead-based flow cytometric analysis and HLA-tetramer analysis.

International Clinical Cytometry Society

ICCS serves the needs of those involved in the current and emerging clinical applications of cytometry throughout the world. It provides information on clinical cases, quality standards, educational resources and an Ask an Expert section.