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Flow cytometry software opens the list-mode data a cytometer writes (almost always FCS files), lets you draw gates, and turns the result into statistics and figures you can save and re-open. This guide follows one workflow, from raw files to a saved analysis, using FlowJo’s documented file handling, then shows a scriptable open-source route and a browser-based option. For the principles behind what you are gating, see how flow cytometry works: dot plots and histograms.
What flow cytometry software is used for in research
Acquisition software on the cytometer records events; analysis software is where you interpret them. In practice that means loading sample files, applying compensation from single-stain controls, gating populations, computing statistics for each gate, and building tables and graphical layouts for a report. FlowJo’s v10 documentation describes this as a workspace that holds loaded samples, statistics, gates, other analyses, and tabular and graphical layouts. Saved as a FlowJo document, reopening it restores the whole analysis.
The same analysis steps apply to conventional and spectral data, and the quality of the result depends on the upstream instrument setup and QC as much as on the package you choose.
Install or access (official source only)
- FlowJo: the official documentation is at docs.flowjo.com/flowjo (checked 2026-10-29). Its Getting Started and Licensing and Installation sections cover obtaining and installing the program, and the Platforms section covers supported systems. Follow those pages rather than a third-party download.
- flowCore (R / Bioconductor): installed from the Bioconductor package page, bioconductor.org/packages/release/bioc/html/flowCore.html (checked 2026-10-29), with the commands shown below.
- Floreada: a browser-based workspace at floreada.io (checked 2026-10-29); nothing is installed.
Step by step: load FCS files and keep the workspace (FlowJo)
These steps follow the FlowJo v10 page on samples and file types (checked 2026-10-29).
- Put the FCS files for the experiment in one folder. FCS is the main format and FlowJo accepts it from nearly all cytometers.
- Open FlowJo so the Workspace window is showing.
- Add the samples in either of two ways: drag the folder of files onto the Workspace window, or click the Add Sample button, then choose the folder and click Choose.
- Check the Samples list in the Workspace window. Samples can be placed in Groups, and a group can be given a role such as compensation or control.
- Draw your gates and compute statistics, using the Graphs and Gating section of the documentation for the current procedure. The question-mark button in any FlowJo window opens the help page for that window.
- Save the workspace. FlowJo opens .WSP and .JO files, which are previously saved analyses, so saving gives you something you can reopen instead of redrawing gates.
- To move an analysis between people or machines, use an .ACS file: it compresses the analysis files, plugin outputs and data into one file, and dragging it into the workspace unpacks it.
What to archive for reproducibility
Keep the original FCS files unchanged, the saved workspace (.WSP) that holds your gates and statistics, and, when sharing, the .ACS bundle. This is the minimum needed for someone else to reopen the same analysis. It fits the habits described in reproducible research practices and belongs in your data management plan.
Output and how to read it
FlowJo’s documentation separates output into two places: Tabular Reports in the Table Editor for statistics per sample and gate, and Graphical Reports in the Layout Editor for figures. Read the table first to confirm population frequencies and counts make sense against your controls, then build the layout from gated plots. Both are documented in the matching sections of docs.flowjo.com.
Troubleshooting
- Samples will not load: confirm the files are FCS, or previously saved analyses (.WSP, .JO) or an .ACS bundle, which are the formats FlowJo’s page lists as openable.
- Gates are gone when you reopen: the analysis only persists if you saved the workspace document; reopen that file, not the raw FCS files.
- Unsure how a window works: use the question-mark button in that window to jump to its help page, or the Technical FAQ section of the documentation.
- Populations look smeared or shifted: check compensation first, using the single-stain control guide.
Open-source and browser alternatives
Scriptable analysis with flowCore
flowCore is a Bioconductor package that, in its own description, provides S4 data structures and basic functions to deal with flow cytometry data. It is licensed Artistic-2.0. Install it from R:
if (!require("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("flowCore")Because the analysis lives in a script, every step can be re-run on the same files.
Comparison of the options covered here
| Tool | How you use it | What its official page states |
|---|---|---|
| FlowJo v10 | Desktop workspace with graphical gating | Workspace holds samples, gates, statistics and layouts; reopening a saved document restores the analysis; opens FCS, .WSP, .JO and .ACS |
| flowCore | R package, script-based | S4 data structures and basic functions for flow cytometry data; Artistic-2.0 license; installed with BiocManager |
| Floreada | Browser-based workspace, no installation | Analysis runs on your device and research data never leaves your computer |
Frequently asked questions
What file format does flow cytometry software read?
FCS is the main format, and FlowJo accepts it from nearly all cytometers. FlowJo also opens saved analyses (.WSP, .JO) and .ACS bundles.
Can I analyse flow cytometry data without installing anything?
Floreada describes itself as a browser-based workspace that runs the analysis on your own device, so the data stays on your computer.
How do I make a flow cytometry analysis reproducible?
Keep the raw FCS files, the saved workspace with its gates, and, for sharing, an .ACS bundle; or run the analysis from a flowCore script.
Where do I learn the exact gating menus?
In the vendor’s current documentation: FlowJo’s Graphs and Gating section, reachable from any window with the question-mark button.
Sources
- FlowJo v10 documentation, docs.flowjo.com/flowjo and Samples and File Types, checked 2026-10-29.
- Bioconductor, flowCore, checked 2026-10-29.
- Floreada, floreada.io, checked 2026-10-29.
