Department of Biomedicine

CyTOF

 Department of Biomedicine CyTOF Home Guidance and support

Guidance and support

This page contains:

>      Links to:   Protocols, User-Guides, Tips/Tricks/WebTool Links
>      Links to:   Data Analysis Software and Toolkits: For SMC and For IMC
>      Links to:   Selected Vendors/Distributors, AU Core Facilities, CyTOF User Groups

Protocols, User-Guides:

Standard BioTools (formally Fluidigm) Protocols and User Guides

SMC: Helios and CYTOF2 Documents ( Appplication Notes, Protocols, Quick References, Software Documents )

IMC:  Hyperion (and Hyperion+) support documents (User Guides, Protocols, Quick References, Software documents)

Selected Published SMC Protocols (Suspension mass cytometry)

Selected Published IMC Protocols (Imaging mass cytometry)

Selected Published Reviews


Tips/Tricks/WebTool Links

Selected Tips/Tricks

WebTools

  • CyTOF Template Creator  Type: Free
    Shiny app for creating a Panel Template for use in CyTOF. (Tested with CyTOF software v. 6.7.1014 and 7.0.8493).
  • CyTOF Panel Designer   (Type: Free; needs registration and password creation at DVSsciences.com)
    Panel Designer v2.0 is available to explore and create new panels with professional advice from their local Field Applications Specialists (from Standard BioTools)
     

Data Analysis Software and Toolkits

For SMC:

Commercial SMC Data Analysis Software:

  • FlowJo (R-Plugins)  Type: Commercial, used at MCU
    Offline Platform for single-cell flow suspension cytometry analysis
  • FCSExpress   Type: Commercial, used at MCU             
    Offline Platform for single-cell suspension cytometry analysis
  • OmiQ  Type: Commercial, not available at MCU
    Online Platform for single-cell suspension cytometry analysis
  • Cytobank    Type: Commercial, not available at MCU
    Online Platform for single-cell suspension cytometry analysis         
  • CellEngine    Type: Commercial, not available at MCU
    Online Platform for single-cell suspension cytometry analysis

Free SMC Data Analysis Software/Scripts/Pipelines:

  • Cyclospore 
    Offline Tool for the analysis of mass cytometry (CyTOF) data
  • Vortex
    VorteX Clustering Environment - Java graphical tool for single-cell analysis, clustering and visualization
    More info from the ClambeyLab: pdf-Link
  • Accense
    Automatic Classification of Cellular Expression by Nonlinear Stochastic Embedding
  • Catalyst 
    R based tool to provide a pipeline for preprocessing of cytometry data, including i) normalization using bead standards, ii) single-cell deconvolution, and iii) bead-based compensation.
  • Spectre
    R package and computational toolkit that enables comprehensive end-to-end integration, exploration, and analysis of high-dimensional cytometry, spatial/imaging, or single-cell data from different batches or experiments
  • Cyanus
    "CYtometry ANalysis Using Shiny": Integrated the methods from the CATALYST package for preprocessing, visualization and clustering. For differential abundance detection, we included the diffcyt methods diffcyt-DA-edgeR, diffcyt-DA-voom and diffcyt-DA-GLMM.
  • CytofKit(2)
    R based Shiny App: An integrated mass cytometry data analysis pipeline that enables simultaneous illustration of cellular diversity and progression.
  • Premessa
    R package for pre-processing of flow and mass cytometry data, that includes panel editing/renaming for FCS files, bead-based normalization and debarcoding.
  • CyTOFPower
    R tool to predict the power of CyTOF experiments in the context of differential state analyses.
    The package provides a shiny app with two options to predict the power of an experiment: i. generation of in-sicilico CyTOF data, using users input ii. browsing in a grid of parameters for which the power was already precomputed.
  • CyCombine
    R-package cyCombine allows for robust integration of single-cell cytometry datasets within and across technologies.
  • Pytometry,  Publication link (2022)
    Flow and mass cytometry analytics in Python.
  • CyTOF workflow (2023)
    Differential discovery in high-throughput high-dimensional cytometry datasets.Workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signaling markers within specific subpopulations, or differential analyses of aggregated signals.
  • CyClone (2023), publication link
    An accessible pipeline to analyze, evaluate, and optimize multiparametric cytometry data (R-script):  
    "Benchmarked and validated Cyclone on mass cytometry (CyTOF), full-spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF)"
  • PICAFlow (2023), publication link
    A complete R workflow dedicated to flow/mass cytometry data, from pre-processing to deep and comprehensive analysis.
  • URSA  (2023), publication link
    An automated multi-omics package for single-cell analysis,
    Ursa is an R package consisting of seven single-cell omics automated analysis workflows. One-liner command for each omics to run a full post-quantification analysis for the omics. Six single-cell (sc) omics and one bulk omics include: scRNA-sequencing (sc), scATAC-sequencing (sc), scImmune profiling (sc), scCNV (sc), CyTOF (sc), Flow cytometry (sc), Spatial transcriptomics (bulk)
  • ImmCellTyper (2023)  publication link
    Cytof data analysis toolkit with cell population auto-annotation function: It has a novel in house semi-supervised clustering tool named BinaryClust, which enables automatic classification and annotation of main cell types as well as in-depth interrogation of certain subpopulation of interest.
  • CyCondor (2024) publication link
    R/Docker based computational framework for the analysis of CyTOF, HDFC, SpectralFlow and CITE-seq data, covering not only all essential steps of cytometry data analysis but also including an array of downstream functions and tools to expand the biological interpretation of the data. The comprehensive suite of features of cyCONDOR, including guided pre-processing, clustering, dimensionality reduction, and machine learning algorithms.

For IMC:

Free IMC Data Analysis Software/Scripts/Pipelines:

Free Standalone Software/Tools/Plugins:

  • MCD viewer  Type: Free
    Post-acquisition data processing software that allows users to visualize, review, and export Imaging Mass Cytometry™ data acquired with the Hyperion™ Imaging System and CyTOF® Software
  • Cell Profiler  Type: Free
    Cell image analysis software
  • histoCAT  Type: Free
    Histology Topography Cytometry Analysis Toolbox (histoCAT) is a package to visualize and analyse multiplexed image cytometry data interactively.
  • ImaCyte  Type: Free
    Interactive tool for data-driven exploration of Imaging Mass Cytometry data
  • Ilastik  Type: Free
    Interactive learning and segmentation toolkit
  • Deep Cell Label (Free)
    DeepCell Label is a data labeling tool to segment images into instance labels, mapping each pixel to an object in the image.
  • Omero  Type: Free
    Open microscopy enviroment for maniging your images
  • 3D Slicer / TITAN Type:Free
                TITAN is an extension developed for 3D Slicer that is responsible for the pre-processing and analysis tasks of imaging mass cytometry (IMC) data
  • Fiji  Type: Free
    Fiji is an image processing package—a “batteries-included” distribution of ImageJ2, bundling a lot of plugins which facilitate scientific image analysis.
  • Mantis-viewer: Image viewer ; Typ: free   MAC/Linux/Windows
    Mantis Viewer is an application developed by the Parker Institute for Cancer Immunotherapy for viewing and analyzing multi-channel pathology imaging, such as IHC, Vectra, MIBI, IMC, CODEX, or other technologies. It has been designed to be highly performant and responsive when analyzing both large and small images and to support workflows with segmentation data.
  • image_utils:  Type: free     Java script
    You can use image-utils to split up unsupported multi-image TIFFs into single channel TIFFs that can be analyzed by Mantis, until  Mantis is supporting this internally.

Free Tools/Pipelines:

  • Multiplexed Image Analysis Tools, developed from the Bodenmiller Lab: Link

    • steinbock for containerized image processing and segmentation.
    • readimc for reading raw IMC data.
    • ImcSegmentationPipeline for interactive IMC data processing and segmentation.
    • napari-imc for interactive visualization of IMC raw data in napari.
    • cytomapper for multiplexed image visualization in R.
    • cytoviewer for interactive multiplexed image visualization in R.
    • imcRtools for single-cell and spatial analysis in R.
    • IMCDataAnalysis for a reproducible workflow on multiplexed image downstream analysis in R.
    • imcdatasets for a collection of publicly available IMC datasets in R.
  • Spectre Type: Free  (https://immunedynamics.io/spectre/#protocols)
    R package and computational toolkit that enables comprehensive end-to-end integration, exploration, and analysis of high-dimensional cytometry, spatial/imaging, or single-cell data from different batches or experiments
  • nfcore/ImCyto  Type: Free
    nfcore/imcyto is a bioinformatics analysis pipeline used for image segmentation and extraction of single cell expression data. The pipeline is built using Nextflow.
    If you want to use it in a windows enviroment, check here: Deploy a complete Nextflow development environment on Windows 10. (Note: May interfear with your windows admin rights)
  • RUNIMC Type: Free
    An R-based package for imaging mass cytometry data analysis and pipeline validation: Publication
  • Cytomapper  Type: Free
    A computational tool written in R, that enables visualization of pixel- and cell-level information obtained by multiplexed imaging. Publication.
    In addition, cytomapper includes a Shiny application that allows hierarchical gating of cells based on marker expression and visualization of selected cells in corresponding images.
  • SIMPLI Type: Free (Linux only?)
    An agnostic pipeline for the analysis of highly multiplexed histological imaging data. SIMPLI performs all the workflow steps: preprocessing, cell-segmentation, phenotyping, as well as spatial and pixel-level analysis.
    SIMPLI is available at: https://github.com/ciccalab/SIMPLI ; Publication
  • "Zaidi"-IMC Pipeline workshop (2022)
    End-to-End Analysis of Imaging Mass Cytometry Data to Study Heterogeneity in Tumor Microenvironments (Pipeline): featuring: QuPath, IMC-converter, StarDist, Python; YouTube Video
    Image dataset, QuPath project, and code are all available here.
  • MCMICRO (Multiple-choice microscopy pipeline) (2022)
    An end-to-end processing pipeline that transforms multi-channel whole-slide images into single-cell data.
    Requires: NextFlow, Docker; Windows: Linux Subssytem or Galaxy enviroment
  • IMC-Denoise (2023).  A content aware denoising pipeline to enhance Imaging Mass Cytometry. Publication
    "IMC-Denoise, to restore IMC images deploying a differential intensity map-based restoration (DIMR) algorithm for removing hot pixels and a self-supervised deep learning algorithm for shot noise image filtering (DeepSNiF). Python/Docker based.
  • PENGUIN (2024).  A rapid and efficient image preprocessing tool for multiplexed spatial proteomics.  Publication
    "PENGUIN's simplicity, speed, and intuitive interface, available as both a script and a Jupyter notebook, make it easy to adjust image processing parameters, providing a user-friendly experience"
    Python/Jupyter based
  • Giotto Suite: a multi-scale and technology-agnostic spatial multi-omics analysis ecosystem (2025): https://www.nature.com/articles/s41592-025-02817-w
    Giotto Suite can be downloaded from our GitHub page github.com/drieslab

  • SPEX (Spatial Expression Explorer): A web-based analysis platform that employs modular analysis pipeline design, accessible through a user-friendly interface. (2025): https://academic.oup.com/gigascience/article/doi/10.1093/gigascience/giaf090/8244097
    SPEX is also available on GitHub: https://github.com/Genentech/spex

     

Commercial IMC data analysis software

  • Halo (Commercia, not available at MCU)
    Image analysis platform for quantitative tissue analysis in digital pathology.
    Related (on demand) webinar: Link
  • Visiopharm (Commercia, not available at MCU)
    image analysis & tissue mining in multi- and highplexed images

Selected CyTOF User groups

AU Links

Selected AU Labs

Selected Vendors/Distributors

Revised 07.01.2026
-
Anders Møller